Coated particles, methods of making and using

ABSTRACT

A particle coated with a nonlamellar material such as a nonlamellar crystalline material, a nonlamellar amorphous material, or a nonlamellar semi-crystalline material includes an internal matrix core having at least one a nanostructured liquid phase, or at least on nanostructured liquid crystalline phase or a combination of the two is used for the delivery of active agents such as pharmaceuticals, nutrients, pesticides, etc. The coated particle can be fabricated by a variety of different techniques where the exterior coating is a nonlamellar material such as a nonlamellar crystalline material, a nonlamellar amorphous material, or a nonlamellar semi-crystalline material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of allowed U.S. patent applicationSer. No. 10/624,498, filed Jul. 23, 2003, which now U.S. Pat. No.6,989,195 is a con of and claims priority to U.S. patent application No.10/170,237, filed Jun. 13, 2002 (now issued U.S. Pat. No. 6,638,621B2),which is a cip and claims priority to U.S. patent application Ser. No.09/297,997, filed Aug. 16, 2000 (now issued U.S. Pat. No. 6,482,517),which is a 371 of International Patent Application PCT/US98/18639, filedSep. 8, 1998, which claims benefit of U.S. provisional patentapplication 60/058,309, filed Sep. 9, 1997. The complete contents of theaforementioned patents and patent applications are hereby incorporatedby reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to coated particles and to methods ofmaking and using them. These coated particles have application in thetargeting and release of one or more materials into selectedenvironments, the absorption of one or more materials from selectedenvironments and the adsorption of one or more materials from selectedenvironments.

2. Related Art

Two particle technologies—polymer-coated particles and liposomes—are ofgeneral interest.

Polymer-coated particles have been very important in the development ofuseful microparticles and of controlled-release vehicles generally. Incertain circumstances polymers have coating and spreading propertiesthat provide for good encapsulation of various matrices, and they areavailable in a range of chemistries and molecular weights. Certainpolymeric coatings are of such utility and low toxicity that approvalhas been obtained for their use even in injectable products within thepharmaceutical industry, most notably polylactic-glycolic acidcopolymers, and the usefulness of polymeric coatings in oral products iswell-established, as in the cases of Eudragits, gelatin, and a number ofnatural gums. In many settings in fact, microparticle coatings aretacitly assumed to be polymers.

However, polymer-coated particles exhibit several limitations, as theflattened and diffuse response of their polymer coatings to chemical andphysical triggers indicates. This is due to two factors. First, the highmolecular weight of polymers reduces their diffusion coefficients andtheir kinetics of solubilization. Second, the neighboring group effectbroadens the curves representing the chemical responses to triggers suchas, inter alia, pH, salinity, oxidation and reduction, ionization, etc.(The neighboring group effect indicates that chemical changes in onemonomeric unit of a polymer significantly alter the parameters governingchemical transitions in each of the neighboring monomeric units.)Further, most polymers are collections of chemical species of broadenedmolecular weight distribution. In addition, for a given application ofthe polymer coated particle only a limited number of suitable polymersare frequently available. This is due to a number of factors: regulatoryissues: the coating processes often entail harsh chemical and/orphysical conditions, such as solvents free radicals, elevatedtemperatures, dessication or drying, and/or macroscopic shearing forcesneeded to form the particles, the limited mechanical and thermalstabilities of the polymeric coatings in industrial applications; andadverse environmental impacts in large scale applications ofpolymer-coated particles, such as in agricultural use.

Liposomes also exhibit a number of limitations. Among these are theirphysical and chemical instabilities. The release of a material disposedwithin the liposome is usually dependent on the destabilization of thestructure of the liposome. In particular, the absence of porosityprecludes the pore-controlled release of such materials. The dualrequirements of 1) physical stability of the liposome until release isdesired on the one hand and 2) release of materials by bilayerdestabilization when release is desired on the other, are problematic.(The term liposomes is frequently interchanged with the term vesiclesand is usually reserved for vesicles of glycerophospholipids or othernatural lipids. Vesicles are self-supported closed bilayer assemblies ofseveral thousand lipid molecules (amphiphiles) that enclose an aqueousinterior volume. The lipid bilayer is a two-dimensional fluid composedof lipids with their hydrophilic head groups exposed to the aqueoussolution and their hydrophobic tails aggregated to exclude water. Thebilayer structure is highly ordered yet dynamic because of the rapidlateral motion of the lipids within the plane of each half of thebilayer.) See O'Brien. D. F. and Rarnaswami, V. (1989) inMark-Bikales-Overbergzer-Menges Encyclopedia of Polymer Science andEngineering. Vol. 17, Ed. John Wiley & Inc., p. 108.

SUMMARY OF THE INVENTION

It is an object of the invention to provide coated particles that aresuitable for solubilizing or containing a wide variety of materials,including materials sensitive to physical, chemical or biologicaldeterioration.

It is an object of the invention to provide coated particles thatrelease one or more material disposed within a matrix in their internalcores without requiring the destabilization of that matrix.

It is an object of the invention to provide coated particles covering awide range of physical and chemical properties, particularly in theselection of the coating, such that a user can substantially preselectthe coating and release characteristics.

It is an object of the invention to provide coated particles thatsharply initiate the release or absorption of one or more materials toor from a selected environment in response to one or more physical orchemical triggers.

It is an object of the invention to provide a wide variety of coatedparticle systems that can be tailored to the particular physical,chemical and biological requirements of their contemplated use, such asmechanical and thermal stability in industrial applications of thecoated particles or freedom from adverse environmental impact in largescale application of the coated particles in agricultural use.

It is an object of the invention to provide coated particles thatprovide, if desired, a porous coating that permits pore-controlledrelease of material disposed within them or pore-controlled absorptionof materials disposed without them.

It is a further object of the invention to provide coated particles thatcan incorporate targeting moieties such as antibodies, lectins,receptors, and complementary nucleic acids, for targeting the particlesto specific sites, either before or after the coating releases, as wellas other bioactive materials such as absorption enhancers, adjuvants,adsorption inhibitors or pharmaceutical actives themselves.

It is a further object of the invention to provide coated particles thatcan be produced by a process that is flexible and can be adapted to awide range of actives, coatings, and matrices.

It is a further object of the invention to provide coated particles thathave a polymerized interior matrix which is more permanent chemically,thermodynamically, and structurally than their unpolymerizedcounterparts.

It is a still further object of the invention to provide coatedparticles that can be made by a simple process, including, preferably,without entailing harsh physical and/or chemical conditions.

The foregoing and other objects are provided by a coated particle thatcomprises an internal core comprising a matrix and an exterior coating.The matrix consists essentially of at least one nanostructured liquidphase, or at least one nanostructured liquid crystalline phase or acombination of the two and the exterior coating comprises a nonlamellarmaterial that is a nonlamellar crystalline material, a nonlamellaramorphous material, or a nonlamellar semi-crystalline material.

In a preferred embodiment, the coated particle may be made by

1. providing a volume of the matrix that includes at least one chemicalspecies having a moiety capable of forming a nonlamellar material uponreaction with a second moiety and

2. contacting the volume with a fluid containing at least one chemicalspecies having the second moiety under nonlamellar solidmaterial-forming conditions so as to react the first moiety with thesecond moiety, and subdividing the volume into particles by theapplication of energy to the volume, or performing this subdivision intoparticles before, and/or after, the chemical reaction.

Alternatively, the coated particle can be made by one of the followingprocesses:

providing a volume of the matrix that includes a material in solution init that is capable of forming a nonlamellar material that is insolublein the matrix and causing the aforesaid material to become insoluble inthe matrix and subdividing the volume into particles by the applicationof energy to the volume;

dispersing particles of said matrix into a fluid that includes at leastone chemical species having a moiety capable of forming a nonlamellarmaterial upon reaction or association with a second moiety and adding tosaid dispersion at least one chemical species having said second moietyto react said first moiety with said second moiety;

dispersing particles of said matrix into a fluid that includes at leastone chemical species having a moiety capable of forming a nonlamellarmaterial upon reaction or association with a second moiety, adding tosaid dispersion at least one chemical species having said second moietyto react said first moiety with said second moiety, and subdividing theresulting material into particles by the application of energy to saidmaterial;

dispersing a volume of said matrix in a form of said nonlamellarmaterial selected from the group consisting of liquefied form, solution,or fluid precursor, and solidifying said nonlamellar material by atechniques selected from the group consisting of cooling, evaporating avolatile solvent, or implementing a chemical reaction; or

dispersing or dissolving a volume of said matrix in a liquid comprisingsaid nonlamellar material in solution or dispersed form and comprisingalso a volatile solvent, and spray-drying said solution or dispersion.

Or, a combination of these methods can be applied.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation, in vertical section, illustrating acoated particle of the present invention comprising an internal corecomprising a 2 by 2 unit cell matrix and an exterior coating:

FIG. 2 is a graphic representation, in section, illustrating a coatedparticle of the present invention;

FIG. 3 is a scanning electron microscope micrograph of coated particlesof the present invention;

FIG. 4 is a scanning electron microscope micrograph of other coatedparticles of the present invention;

FIG. 5 is a graph of the measured volume-weighted cumulative particlesize distribution for coated particles of the present invention on avolume-weighted particle diameter versus cumulative particle size basis;

FIG. 6 is a graph of measured small-angle X-ray scattering intensityversus wave vector q of coated particles of the present invention;

FIG. 7 is a graph of detector counts versus elution time in minutes fora control using high pressure liquid chromatography; and

FIG. 8 is a graph of detector counts versus elution time in minutes forcoated particles of the present invention using high pressure liquidchromatography.

FIG. 9. A phase-contrast optical micrograph of PLGA-coatedmicroparticles dispersed in water, showing the core-shell structure.

FIG. 10. On the left is a PLGA-coated cubic phase, made according to theinstant invention, soaking in linalool, which is a non-solvent for PLGAbut a solvent for the cubic phase. On the right, the same cubic phasewas soaked in linalool under identical conditions, demonstrating thatthe cubic phase dissolves in the linalool when not coated.

FIG. 11. A large (5 mm) particle of coated cubic phase in which thecoating consists of amorphous trehalose, obtained by freeze-drying adispersion of Arlatone G-based cubic phase in a trehalose solution.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As illustrated in FIGS. 1 and 2, coated particle 1 used in the presentinvention comprises an internal core 10 and a coating 20 exterior to it(hereinafter “exterior coating 20”). The internal core 10 comprises amatrix consisting essentially of a nanostructured material selected fromthe group consisting of

-   -   a. at least one nanostructured liquid phase.    -   b. at least one nanostructured liquid crystalline phase and    -   c. a combination of        -   i. at least one nanostructured liquid phase and        -   ii. at least one nanostructured liquid crystalline phase.            Alternatively, the interior could be a composition that            yields one of these phases upon contact with water or other            aqueous fluid.

The liquid phase material and the liquid crystalline phase material mayeither contain solvent (lyotropic) or not contain solvent(thermotropic). The exterior coating 20 comprises a nonlamellarmaterial. The term “exterior coating” as used herein is intended toindicate that the coating 20 is exterior to the internal core 10 and isnot intended to be limited to meaning that the exterior coating 20 isthe most exterior coating of the coated particle 1. For instance, inmany of the Examples given herein, a surfactant-rich layer is present atthe outer surface of the non-lamellar exterior coating. And in otherembodiments presented, an antibody or other bioactive material will beadsorbed to, or extending out from, this non-lamellar exterior coating.

Nanostructured liquid phase and nanostructured liquid crystalline phasepossess unique properties that are not only important in making possiblethe easy production of particles according to the present invention, butalso yield highly desirable solubilization, stability, and presentationproperties and other capabilities in the final coated particles of thepresent invention.

As for the exterior coating 20, non-lamellar structures which exhibitbonding and/or packing rigidity that extends in all three dimensions arestrongly preferred in the present invention over lamellar materials, dueto the well-known physical and chemical limitations and instabilities oflamellar, and more generally layered, structures, as exemplified by, forexample, (a) the instability (even when acquiescent) of emulsions whichhave droplets coated with lamellar liquid crystalline layers, (b) thechemical instability upon removal of guest molecules in certain Wemercomplexes, and (c) the dramatically inferior hardness and shear modulusfor graphite as compared with diamond.

Coated particles 1 used in the present invention may be from 0.1 micronto 30 microns or above in mean caliper diameter, and preferably fromabout 0.2 micron to about 5 micron in mean caliper diameter. Macroscopicparticles can be made as well, i.e., particles with sizes measured inmillimeters or even larger, as exemplified in Examples 39 and 40; theability to make particles of this larger size could open up applicationsof the present invention in, for example, depot delivery systems forsustained release upon implantation. The coated particle 1 used may alsobe provided with a stabilizing layer on its exterior, i.e. outside theexterior coating 20 as desired, such as a polyelectrolyte or surfactantmonolayer to prevent agglomeration of coated particles 1.

The coated particles 1 used in the present invention have application ina variety of modalities of use. The coated particle 1 may, upon releaseof the exterior coating 20, absorb one or more materials from a selectedenvironment, adsorb one or more materials from a selected environment orrelease one or more materials, such as active agents disposed in thematrix, into a selected environment, and/or target specific sites forthe intended release or ad/absorption. Alternatively, certain exteriorcoatings possessing porosity, such as inclusion compounds and zeolites,do not require release in order to effect the absorption or release of amaterial of interest into or out of the matrix, and in some such casesvery high selectivity can be obtained by the use of properly tuned porecharacteristics. In cases where the particles are used to adsorb acompound or compounds of interest, neither porosity nor release of theexterior coating 20 are required, but porosity can provide for a verylarge increase in adsorption capacity by allowing the adsorbed materialto diffuse into the matrix, making the adsorption sites in the exteriorcoating 20 available to adsorb new material. In a preferred embodiment,an additional material, such as an active agent, may be disposed withinthe matrix for release into a selected environment.

Coating: In the present context of particles, a “coating” is composed ofa material which behaves as a solid in the common sense, and in theengineering viewpoint, of the term “solid”, namely that it exhibits arigidity and permanence that contrasts sharply with low-viscosityliquids, and thus represents a significant diffusional barrier to thepassage of compounds across that material, in a way that is intuitivelydifferent from any protection that a low-viscosity liquid layer couldprovide. This common sense understanding of the terms “liquid” and“solid” differs fundamentally from the strict scientific definitions,which refer only to the existence or non-existence of long-range atomicorder. Thus, while an amorphous material such as PMMA (Plexiglass) orordinary glass—particles of which make up an everyday coating known asceramic glaze—may technically be a liquid, for the purposes ofsimplifying nomenclature in the context of this invention thesematerials will be referred to as solids, as they would be in ordinarylife outside of the physics laboratory.

-   -   The matrix is    -   a. thermodynamically stable    -   b. nanostructured and    -   c. a liquid phase or liquid crystalline phase or a combination        thereof.        Nanostructured: The terms “nanostructure” or “nanostructured” as        used herein in the context of the structure of a material refer        to materials the building blocks of which have a size that is on        the order of nanometers (10⁻⁹ meter) or tens of nanometers        (10×10⁻⁹ meter). Generally speaking, any material that contains        domains or particles 1 to 100 nm (nanometers) across, or layers        or filaments of that thickness, can be considered a        nanostructured material. (See also Dagani, R., “Nanostructured        Materials Promise to Advance Range of Technologies.” Nov. 23,        1992 C&E News 18 (1992).) The term is meant to exclude so-called        “ceramic glasses” which are crystalline materials in which the        crystallite size is so small that one may not observe peaks in        wide-angle x-ray diffraction and which some physicists may refer        to as nanostructured materials: the nanostructured liquid and        liquid crystalline phases that are defined herein are        characterized by nanoscale domains which are clearly        distinguished from neighboring domains by large differences in        local chemical composition, and do not include materials in        which neighboring domains have essentially the same local        chemical composition and differ only in lattice orientation.        Thus, by the term ‘domain’ as used herein it is meant a spatial        region which is characterized by a particular chemical makeup        which is clearly distinguishable from that of neighboring        domains: often such a domain is hydrophilic (hydrophobic) which        contrasts with the hydrophobicity (hydrophilicity) of        neighboring domains: in the context of this invention the        characteristic size of these domains is in the nanometer range.        (The term ‘microdomain’ is often used to indicate domains whose        size range is micron or nanometer scale.)        Nanostructured liquids and liquid crystals: Nanostructured        liquid phases and liquid crystalline phases, which provide the        matrix of the internal cores 10 of the coated particles 1 in the        present invention, possess unique collections of properties that        are not only crucial in making possible the production of        particles of the present invention, but also yields highly        desirable solubilization, stability, and presentation properties        and capabilities in the final coated particles. As discussed in        more detail below in the discussion of particle production        processes, in order that a material provide for ready        dispersibility with one of the processes described herein, it is        desirable for the material to be of very low solubility in water        (otherwise it will tend to dissolve during the dispersing        process, limiting dispersibility), yet, at the same time it        should contain water—both for the purpose of solubilizing        water-soluble reactants used in dispersing and for making        possible the solubilization of a large range of active        compounds.

In particular, for solubilization of hydrophilic (especially charged)and amphiphilic compounds, and for the maintenance of not onlysolubilization but also proper conformation and activity, of sensitivecompounds of biological origin such as proteins, the interior matrixshould contain substantial concentrations of water or other polarsolvent. In terms of establishing versatility in coating selection, agreat many (perhaps a majority) of the compounds listed as usefulcoatings in the present invention require reactants that are solubleonly in polar solvents. Furthermore, the use of organic solvents forsolubilization is in most cases inconsistent with the present matricesand/or with active biological compounds such as proteins (used in thepresent invention as actives or as targeting agents), and in any case ishighly disfavored from regulatory, environmental, and healthconsiderations. These two requirements of water-insolubility andsolubilization of water-soluble compounds are, of course, working inopposite directions and are difficult to resolve in a single,inexpensive, and safe material.

Very effective systems for satisfying such solubilization requirementsare provided by lipid-water systems, in which microdomains are presentwhich are very high in water content, and simultaneously hydrophobicdomains are in very close contact with the aqueous domains. The presenceof aqueous domains circumvents precipitation tendencies encountered insystems where water structure is interrupted by the presence of highloadings of co-solvents or co-solutes, as, for example, in concentratedaqueous polymer solutions. At the same time the proximity of hydrophobicdomains provides for effective solubilization of amphiphilic compounds(and hydrophobic as well).

Nanostructured liquid and liquid crystalline phases are synthetic orsemisynthetic materials which adopt these solubilizationcharacteristics, and provide pure, well-characterized, easily produced,and typically inexpensive matrices that also have the followingdesirable properties:

a) versatility in chemical systems forming nanostructured liquid phasesand nanostructured liquid crystalline phases, ranging from biologicallipids that are ideal for biomolecules, to hardy fluorosurfactants, toglycolipids that bind bacteria, to surfactants with ionic or reactivegroups, etc. This provides for applicability over a wide range ofconditions and uses;

b) the unsurpassed ability of nanostructured liquid phases andnanostructured liquid crystalline phases to: i) solubilize a wide rangeof active compounds including many traditionally difficult compoundssuch as Paclitaxel and biopharmaceuticals, circumventing the need fortoxic and increasingly regulated organic solvents; ii) achieve highconcentrations of actives with uncompromised stability, and iii) providethe biochemical environment that preserves their structure and function.

c) true thermodynamic stability, which greatly reduces instabilitiescommon with other vehicles, such as precipitation of active agents,breaking of emulsions, vesicle fusion, etc., and,

d) the presence of a porespace with preselectable pore size in thenanometer range, facilitating further control of the release kineticseven after triggered release of the coating, particularly in the releaseof proteins and other biomacromolecules.

The desired properties of the nanostructured material of the internalcore 10 derive from several related concepts regarding materials thatcan be described with respect to surfactants by use of the terms“polar,” “apolar,” “amphiphile” “surfactant,” and the “polar-apolarinterface, and analogously with respect to block copolymer systems, asdescribed below.

Polar: polar compounds (such as water) and polar moieties (such as thecharged head groups on ionic surfactants or on lipids) are water-lovingor hydrophilic “polar” and “hydrophilic” in the context of the presentinvention are essentially synonymous. In terms of solvents, water is notthe only polar solvent. Others of importance in the context of thepresent Invention are: glycerol, ethylene glycol, formamide, N-methylformamide, dimethylformamide, ethylammonium nitrate, acetamide,N-methylacetamide, dimethylacetamide, N-methyl sydnone, and polyethyleneglycol. Note that one of these (polyethylene glycol) is actually apolymer, thereby illustrating the range of possibilities. Atsufficiently low molecular weights, polyethylene glycol (PEG) is aliquid, and although PEG has not been extensively studied as a polarsolvent in combination with surfactants, it has been found that PEG doesform nanostructured liquid phases and liquid crystalline phases incombination with, for example, surfactants such as BRIJ-typesurfactants, which are nonionic surfactants with PEG head groupsether-linked to alkane chains. More generally, in terms of polar groupsin hydrophilic and amphiphilic molecules (including but not limited topolar solvents and surfactants), a number of polar groups are tabulatedbelow, in the discussion of which polar groups are operative assurfactant head groups and which are not.Apolar. An apolar compound is a compound that has no dominant polargroup. Apolar (or hydrophobic, or alternatively, “lipophilic”) compoundsinclude not only the paraffinic/hydrocarbon/alkane chains ofsurfactants, but also modifications of them, such as perfluorinatedalkanes, as well as other hydrophobic groups such as the fused-ringstructure in cholic acid as found in bile salt surfactants, or phenylgroups as form a portion of the apolar group in Triton-type surfactants,and oligomer and polymer chains that run the gamut from polyethylene(which represents a long alkane chain) to hydrophobic polymers such ashydrophobic polypeptide chains in novel peptide-based surfactants thathave been investigated. A listing of some apolar groups and compounds isgiven below, in the discussion of useful components of thenanostructured phase interior. An apolar compound will be lacking inpolar groups, a tabulation of which is included herein, and willgenerally have an octanol-water partition coefficient greater than about100, and usually greater than about 1,000.Amphiphile: an amphiphile can be defined as a compound that containsboth a hydrophilic and a lipophilic group. See D. H. Everett, Pure andApplied Chemistry, vol. 31, no. 6. p. 611, 1972. It is important to notethat not every amphiphile is a surfactant. For example, butanol is anamphiphile, since the butyl group is lipophilic and the hydroxyl grouphydrophilic, but it is not a surfactant since it does not satisfy thedefinition, given below. There exist a great many amphiphilic moleculespossessing functional groups which are highly polar and hydrated to ameasurable degree, yet which fail to display surfactant behavior. See R.Laughlin. Advances in liquid crystals, vol. 3, p. 41, 1978.Surfactant: A surfactant is an amphiphile that possesses two additionalproperties. First, it significantly modifies the interfacial physics ofthe aqueous phase (at not only the air-water but also the oil-water andsolid-water interfaces) at unusually low concentrations compared tononsurfactants. Second, surfactant molecules associate reversibly witheach other (and with numerous other molecules) to a highly exaggerateddegree to form thermodynamically stable, macroscopically one-phase,solutions of aggregates or micelles. Micelles are typically composed ofmany surfactant molecules (10's to 1000's) and possess colloidaldimensions. See R. Laughlin, Advances in liquid crystals, vol. 3, p. 41,1978. Lipids and polar lipids in particular, often are considered assurfactants for the purposes of discussion herein, although the term‘lipid’ is normally used to indicate that they belong to a subclass ofsurfactants which have slightly different characteristics than compoundswhich are normally called surfactants in everyday discussion. Twocharacteristics which frequently, though not always, are possessed bylipids are first, they are often of biological origin, and second, theytend to be more soluble in oils and fats than in water. Indeed, manycompounds referred to as lipids have extremely low solubilities inwater, and thus the presence of a hydrophobic solvent may be necessaryin order for the interfacial tension-reducing properties and reversibleself-association to be most clearly evidenced, for lipids which areindeed surfactants. Thus, for example, such a compound will stronglyreduce the interfacial tension between oil and water at lowconcentrations, even though extremely low solubility in water might makeobservation of surface tension reduction in the aqueous systemdifficult. Similarly, the addition of a hydrophobic solvent to alipid-water system might make the determination of self-association intonanostructured liquid phases and nanostructured liquid crystallinephases a much simpler matter, whereas difficulties associated with hightemperatures might make this difficult in the lipid-water system.

Indeed, it has been in the study of nanostructured liquid crystallinestructures that the commonality between what had previously beenconsidered intrinsically different—‘lipids’ and ‘surfactants’—came tothe forefront, and the two schools of study (lipids, coming from thebiological side, and surfactants, coming from the more industrial side)came together as the same nanostructures were observed in lipids as forall surfactants. In addition, it also came to the forefront that certainsynthetic surfactants, such as dihexadecyldimethylammonium bromide,which were entirely of synthetic, non-biological origin, showed‘lipid-like’ behavior in that hydrophobic solvents were needed forconvenient demonstration of their surfactancy. On the other end, certainlipids such as lysolipids, which are clearly of biological origin,display phase behavior more or less typical of water-solublesurfactants. Eventually, it became clear that for purposes of discussingand comparing self-association and interfacial tension-reducingproperties, a more meaningful distinction was between single-tailed anddouble-tailed compounds, where single-tailed generally implieswater-soluble and double-tailed generally oil soluble.

Thus, in the present context, any amphiphile which at very lowconcentrations lowers interfacial tensions between water and hydrophobe,whether the hydrophobe be air or oil, and which exhibits reversibleself-association into nanostructured micellar, inverted micellar, orbicontinuous morphologies in water or oil or both, is a surfactant. Theclass of lipids simply includes a subclass consisting of surfactantswhich are of biological origin.

Polar-apolar interface: In a surfactant molecule, one can find adividing point (or in some cases two points, if there are polar groupsat each end, or even more than two, as in Lipid A, which has seven acylchains and thus seven dividing points per molecule), in the moleculethat divide the polar part of the molecule from the apolar part. In anynanostructured liquid phase or nanostructured liquid crystalline phase,the surfactant forms monolayer or bilayer films: in such a film, thelocus of the dividing points of the molecules describes a surface thatdivides polar domains from apolar domains: this is called the“polar-apolar interface” or “polar-apolar dividing surface.” Forexample, in the case of a spherical micelle, this surface would beapproximated by a sphere lying inside the outer surface of the micelle,with the polar groups of the surfactant molecules outside the surfaceand apolar chains inside it. Care should be taken not to confuse thismicroscopic interface with macroscopic interfaces separating two bulkphases that are seen by the naked eye.Bicontinuous: In a bicontinuous structure, the geometry is described bytwo distinct, multiply-connected, intertwined subspaces each of which iscontinuous in all three dimensions: thus, it is possible to traverse theentire span of this space in any direction even if the path isrestricted to one or other of the two subspaces. In a bicontinuousstructure, each of the subspaces is rich in one type of material ormoiety, and the two subspaces are occupied by two such materials ormoieties each of which extends throughout the space in all threedimensions. Sponge, sandstone, apple, and many sinters are examples ofrelatively permanent though chaotic bicontinuous structures in thematerial realm. In these particular examples, one of the subspaces isoccupied by a solid that is more or less deformable and the othersubspace, though it may be referred to as void, is occupied by a fluid.Certain lyotropic liquid crystalline states are also examples, onesubspace being occupied by amphiphile molecules oriented and aggregatedinto sheet-like arrays that are ordered geometrically, the othersubspace being occupied by solvent molecules. Related liquid crystallinestates that contain two incompatible kinds of solvent molecules, e.g.hydrocarbon and water, present a further possibility in which onesubspace is rich in the first solvent, the other in the second, and thesurface between lies within a multiply connected stratum rich inoriented surfactant molecules. Certain equilibrium microemulsion phasesthat contain comparable amounts of hydrocarbon and water as well asamphiphilic surfactant may be chaotic bicontinuous structures,maintained in a permanent state of fluctuating disorder by thermalmotions, for they give no evidence of geometric order but there iscompelling evidence for multiple continuity. Bicontinuous morphologiesoccur also in certain phase-segregated block copolymers. See Anderson,D. M., Davis, H. T., Nitsche, J. C. C. and Scriven, L. E. (1900)Advances in Chemical Physics, 77:337.Chemical criteria: A number of criteria have been tabulated anddiscussed in detail by Robert Laughlin for determining whether a givenpolar group is functional as a surfactant head group, where thedefinition of surfactant includes the formation in water ofnanostructured phases even at rather low concentrations. R. Laughlin,Advances in Liquid Crystals, pp. 3–41, 1978.

The following listing given by Laughlin gives some polar groups whichare not operative as surfactant head groups—and thus, for example, analkane chain linked to one of these polar groups would not be expectedto form nanostructured liquid or liquid crystalline phases—are:aldehyde, ketone, carboxylic ester, carboxylic acid, isocyanate, amide,acyl cyanoguanidine, acyl guanyl urea, acyl biuret, N,N-dimethylamide,nitrosoalkane, nitroalkane, nitrate ester, nitrite ester, nitrone,nitrosamine, pyridine N-oxide, nitrile, isonitrile, amine borane, aminehaloborane, sulfone, phosphine sulfide, arsine sulfide, sulfonamide,sulfonamide methylimine, alcohol (monofunctional), ester(monofunctional), secondary amine, tertiary amine, mercaptan, thioether,primary phosphine, secondary phosphine, and tertiary phospine.

Some polar groups which are operative as surfactant head groups, andthus for example, an alkane chain linked to one of these polar groupswould be expected to form nanostructured liquid and liquid crystallinephases, are:

-   -   a. Anionics: carboxylate (soap), sulfate, sulfamate, sulfonate,        thiosulfate, sulfinate, phosphate, phosphonate, phosphinate,        nitroamide, tris(alkylsulfonyl)methide, xanthate;    -   b. Cationics: ammonium, pyridinium, phosphonium, sulfonium,        sulfoxonium;    -   c. Zwiterionics: ammonio acetate, phosphoniopropane sulfonate,        pyridinioethyl sulfate;    -   d. Semipolars: amine oxide, phosphonyl, phosphine oxide, arsine        oxide, sulfoxide, sulfoximine, sulfone diimine, ammonio amidate.

Laughlin also demonstrates that as a general rule, if the enthalpy offormation of a 1:1 association complex of a given polar group withphenol (a hydrogen bonding donor) is less than 5 kcal, then the polargroup will not be operative as a surfactant head group.

In addition to the polar head group, a surfactant requires an apolargroup, and again there are guidelines for an effective apolar group. Foralkane chains, which are of course the most common, if n is the numberof carbons, then n must be at least 6 for surfactant associationbehavior to occur, although at least 8 or 10 is the usual case.Interestingly octylamine, with n=8 and the amine head group which isjust polar enough to be effective as a head group, exhibits a lamellarphase with water at ambient temperature, as well as a nanostructured L2phase. Warnhelm, T., Bergenstahl, B., Henriksson, U., Malmvik, A. -C.and Nilsson, P. (1987) J. of Colloid and Interface Sci. 118:233.Branched hydrocarbons yield basically the same requirement on the low nend; for example, sodium 2-ethylhexylsulfate exhibits a full range ofliquid crystalline phrases. Winsor, P. A. (1968) Chem. Rev. 68:1.However, the two cases of linear and branched hydrocarbons are vastlydifferent on the high n side. With linear, saturated alkane chains, thetendency to crystallize is such that for n greater than about 18, theKrafft temperature becomes high and the temperature range ofnanostructured liquid and liquid crystalline phases increases to hightemperatures, near or exceeding 100° C. In the context of the presentinvention, for most applications this renders these surfactantsconsiderably less useful than those with n between 8 and 18. With theintroduction of unsaturation or branching in the chains, the range of ncan increase dramatically. The case of unsaturation can be illustratedwith the case of lipids derived from fish oils, where chains with 22carbons can have extremely low melting points due to the presence of asmany as 6 double bonds, as in docosahexadienoic acid and itsderivatives, which include monoglycerides, soaps, etc. Furthermore,polybutadiene of very high MW is an elastomeric polymer at ambienttemperature, and block copolymers with polybutadiene blocks are wellknown to yield nanostructured liquid crystals. Similarly, with theintroduction of branching one can produce hydrocarbon polymers such aspolypropyleneoxide (PPO) which serves as the hydrophobic block in anumber of amphiphilic block copolymer surfactants of great importance,such as the Pluronic series of surfactants. Substitution of fluorine forhydrogen, in particular the use of perfluorinated chains, in surfactantsgenerally lowers the requirement on the minimal value of n, asexemplified by lithium perfluourooctanoate (n=8), which displays a fullrange of liquid crystalline phases, including an intermediate phasewhich is fairly rare in surfactant systems. As discussed elsewhere,other hydrophobic groups, such as the fused-ring structure in thecholate soaps (bile salts), also serve as effective apolar groups,although such cases must generally be treated on a case by case basis interms of determining whether a particular hydrophobic group will yieldsurfactant behavior.

For single-component block copolymers, relatively simple mean-fieldstatistical theories are sufficient to predict when nanostructure liquidphase and liquid crystalline phase materials will occur and these arequite general over a wide range of block copolymers. If χ is theFloryHuzuins interaction parameter between polymer blocks A and B, and Nis the total index of polymerization defined as the number ofstatistical units or monomer units in the polymer chain, consistentlywith the definition of the interaction parameter of the block copolymer,then nanostructure liquid and liquid crystalline phases are expectedwhen the product χ N is greater than 10.5, Leibler, L. (1980)Macromolecules 13:1602. For values comparable to but larger than thiscritical value of 10.5, ordered nanostructured (liquid crystalline)phases can occur, including ever bicontinuous cubic phases. Hajduk, D.A., Harper, P. E., Gruner, S. M., Honeker, C. C., Kim, G., Thomas, E. L.and Fetters, L. J. (1994) Macromolecules 27:4063.

The Nanostructured Liquid Phases of Utility

The nanostructured liquid phase material suitable for the nanostructuredmaterial of the matrix may be

a. a nanostructured L1 phase material,

b. a nanostructured L2 phase material,

c. a nanostructured microemulsion or

d. a nanostructured L3 phase material.

The nanostructured liquid phases are characterized by domain structurescomposed of domains of at least a first type and a second type (and insome cases three or even more types) having the following properties:

a) the chemical moieties in the first type domains are incompatible withthose in the second type domains (and in general, each pair of differentdomain types are mutually incompatible) such that they do not mix underthe given conditions but rather remain as separate domains; for example,the first type domains could be composed substantially of polar moietiessuch as water and lipid head groups, while the second type domains couldbe composed substantially of apolar moieties such as hydrocarbon chains:or, first type domains could be polystyrene-rich, while second typedomains are polyisoprene-rich and third type domains arepoly-vinylpyrrolidone-rich;

b) the atomic ordering within each domain is liquid-like rather thansolid-like, i.e., it lacks lattice-ordering of the atoms; this would beevidenced by an absence of sharp Bragg peak reflections in wide-anglex-ray diffraction;

c) the smallest dimension (e.g., thickness in the case of layers,diameter in the case of cylinder-like or sphere-like domains) ofsubstantially all domains is in the range of nanometers (viz., fromabout 1 to about 100 nm); and

d) the organization of the domains does nor exhibit long-range order norconform to any periodic lattice. This is evidenced by the absence ofsharp Bragg reflections in small-angle x-ray scattering examination ofthe phase. Furthermore, as seen below, if high viscosity andbirefringence are both lacking, this is strong evidence of a liquid, asopposed to liquid crystalline, phase.

With respect to each of the liquid phases, systems based on surfactants,where the two types of domains in the nanostructured liquid are ‘polar’and ‘apolar’ are initially discussed. Generally, following that, systemsbased on block copolymers are discussed. In these systems the terms‘polar’ and ‘apolar’ may or may not be applicable, but there existdomain types ‘A’, ‘B’, etc. where as defined above (in the definition ofa nanostructure liquid) domain types ‘A’ and ‘B’ are immiscible withrespect to each other.

L1 phase: In an L1 phase that occurs in a system based on surfactants,the curvature of the polar-apolar interface is toward the apolar(non-polar) regions, generally resulting in particles—normalmicelles—that exist in a water-continuous medium. (Here “water” refersto any polar solvent). When these micelles transform from spherical tocylindrical as conditions or compositions change, they can start to fusetogether and bicontinuity can result. In addition to the watercontinuity, the hydrophobic domains can connect up to form asample-spanning network; this can still be an L1 phase. In addition,there are examples of L1 phases that show evidence of having nomicrostructure whatsoever. That is, there are no micelles, nowell-defined domains, just surfactant molecules co-mingled in astructureless, one-phase liquid solution that is thus not ananostructured material. These “structureless solutions” can sometimesbe changed to nanostructured phases by simple change in compositionwithout any phase change in between. In other words, thermodynamics doesnot dictate a phase boundary between a structureless solution and ananostructured phase. This is, of course, in contrast with the case of atransition between a phase having long-range order (a liquid crystal ora crystal) and a phase lacking long-range order (a liquid), where aphase boundary is required by thermodynamics.

For L1 phases that occur in systems based on block copolymers, the terms‘polar’ and apolar may not apply, but in any case there are two (or insome cases more) domain types; we make the convention that the curvatureof the A/B interface is toward A domains, so that a typicalnanostructure should consist of particles, often sphere-like, of domaintype A located in a continuum of B domains. As an example, inpolystyrene-polyisoprene diblock copolymers, if the volume fraction ofpolystyrene blocks is very low, say 10%, then the usual microstructurewill be polystyrene-rich spheres in a continuous polyisoprene matrix.Contrariwise, polyisoprene-rich spheres in a polystyrene-continuousmatrix would be the likely structure for a 10% polyisoprene PS-PIdiblock.

Identification of the nanostructured L1 phase. Since the L1 phase is aliquid phase, techniques have been developed to distinguished thenanostructured L1 phase from unstructured solution liquid phases. Inaddition to the experimental probes that are discussed below, there is awell-known body of knowledge that provides criteria by which one candetermine a priori whether a given system should be expected to formnanostructured phases instead of simple unstructured solutions.

Since the formation of nanostructured liquid phases and nanostructuredliquid crystalline phases is one requirement in the definition of asurfactant, in the discrimination of a nanostructured liquid from anunstructured solution it is extremely valuable to have criteria fordetermining whether a given compound is in fact a surfactant, criteriawhich provide for a number of tests for surfactancy in addition tomethods discussed below for directly analyzing the liquid in question. Anumber of criteria have been discussed by Robert Laughlin in Advances inLiquid Crystals, 3:41, 1978. To begin with, Laughlin lists chemicalcriteria for determining a priori whether a given compound will be asurfactant, and this was discussed in detail above. If, based on thesecriteria, a compound is expected to be a true surfactant, then thecompound is expected to form nanostructured phases in water. Inaddition, with such a compound in the presence of water and hydrophobe,nanostructured phases are also expected to form normally, incorporatingat least a portion of the hydrophobe present.

In the event that a non-surfactant amphiphile is added to such a system,and in particular an amphiphilic organic solvent such as a short-chainedalcohol, dioxane, tetrahydrofuran, dimethylformamide, acetonitrile,dimethylsulfoxide, etc., then structureless liquids could form as theaction of the organic solvent will generally be to disrupt colloidalaggregates and cosolubilize all the components.

Laughlin also goes on to discuss a number of criteria based on physicalobservations. One well-known criteria is the critical micelleconcentration (CMC) which is observed in surface tension measurements.If the surface tension of an aqueous solution of the compound inquestion is plotted as a function of the concentration, then at very lowconcentrations, the surface tension will be seen to drop off sharply ifthe added compound is indeed a surfactant. Then, at a particularconcentration known as the CMC, a sharp break will occur in this plot asthe slope of the line decreases drastically, to the right of the CMC, sothat the surface tension decreases much less with added surfactant. Thereason is that above the CMC, added surfactant goes almost entirely intothe creation of micelles rather than to the air-water interface.

A second criterion tabulated by Laughlin is the liquid crystalcriterion: if the compound forms liquid crystals at high concentrations,then it must be a surfactant and will form liquid crystalline phases atconcentrations lower than those at which the occur. In particular, theL1 phase is usually found at concentrations of surfactant just lowerthan those that form normal hexagonal, or in some cases normalnon-bicontinuous cubic phase liquid crystals.

Another criterion discussed by Laughlin is based on the temperaturedifferential between the upper limit of the Krafft boundary plateau andthe melting point of the anhydrous compound. The Krafft boundary is acurve in the phase diagram of the binary system with compound and water:below he Krafft line are crystals—and above the Krafft line the crystalsmelt, so that there is a dramatic increase in solubility over a verynarrow temperature range along the Krafft line. In the case of a truesurfactant, this temperature differential is substantial: for example,in sodium palmitate, the melting point of the anhydrous compound is 288°C., while the Krafft line has its plateau at 69° C., so that thedifferential is 219° C. Laughlin goes on to discuss the case ofdodecylamine, which has a temperature differential of 14° C., and has asmall region in the phase diagram corresponding to liquid crystals, thusindicating a modest degree of association colloid behavior. In contrast,neither dodecylmethylamine nor dodecanol exhibit association behavior ofthe surfactant type, and both have zero temperature differential.

As in the case of liquid crystals, as discussed herein, given a materialthere are a number of experimental probes one can use to determinewhether or not the material, in this case a liquid, is nanostructured,and these will be discussed in the context of the L1 phase, althoughthey apply to all nanostructured liquids, with the appropriatemodifications. In such a determination, it is best to combine as many ofthese characterizations as feasible.

As with all the liquid phases, the L1 phase is optically isotropic inthe absence of flow. It does not give a splitting in the ²H NMRbandshape with deuterated surfactant.

Also, in examination with crossed polarizing filters, the L1 phase ofsurfactant systems does not generally give birefringence even undermoderate flow conditions. The situation with respect to birefringence inthe case of block copolymer-based systems is complicated by thepossibility of strain birefringence, so this is not a reliable method inthat case.

Returning to the surfactant-based L1 phase, viscosity is generally quitelow, considerably lower than in liquid crystals in the same system.

Using pulsed-gradient NMR to measure the effective self-diffusioncoefficients of the various components, one finds that theself-diffusion of surfactant, and any added hydrophobe is very low,typically on the order of 10⁻¹³ m²/sec or less (unless the phase isbicontinuous: see below). This is because the primary means fordiffusion of surfactant and hydrophobe is by diffusion of entiremicelles, which is very slow. Also, the diffusion rates of surfactantand of hydrophobe should be nearly the same, for the same reason.

Small-angle x-ray scattering (SAXS) does not give sharp Bragg peaks inthe nanometer range (nor any range), of course. However, analysis of theentire curve by several methods from the literature can give the lengthscale of the nanostructure. By analyzing the falloff of intensity at lowwave numbers (but not too low compared to the inverse of the surfactantmolecule length), one can determine the apparent radius of gyration: oneplots intensity, versus the square of the wave number, and takes theslope to deduce R_(g) (the so-called Guinier plot). The radius ofgyration is then related to the dimensions of the micellar units bystandard well-known formulae. This will fall in the range of nanometers.In addition, by plotting the product of intensity times the square ofthe wave number versus the wave number—the so-called ‘Hosemann plot’—onewill find a peak that can also be related to the dimensions of themicelles; this has the advantage that it is less sensitive tointeractions between micelles than is the radius of gyration.

For surfactant-based L1 phases which are bicontinuous, the above willchange as follows: First, the viscosity can increase considerably whenbicontinuity occurs, do to the rigidity of the surfactant film, which iscontinuous. Also, the self-diffusion rate of the surfactant and even ofadded hydrophobe which can be deliberately added to a binary system as amarker) can increase dramatically, approaching or even exceeding thevalues in a lamellar phase in the same system. And while SAXS analyses,both the radius of gyration and the Hosemann plot, will give resulting,dimensions in the nanometer range, these must be interpreted ascharacteristic length scales of the bicontinuous domain structure,rather than as dimensions of discrete particles. In some models, such asthe interconnected cylinders model of the author's thesis, or theTalmon-Prager model, a bicontinuous domain structure is represented asmade up of units which although seemingly ‘particles’ are in realityonly building blocks for construction of a model bicontinuous geometry.

For L1 phases in block copolymer-based systems, this same SAXS analysisholds. In contrast, NMR bandshape and self-diffusion measurements ingeneral do not carry over, nor do surface tension measurements. However,vapor transport measurements have been used in the past in place of NMRself-diffusion, in particular, if one can find a gas which ispreferentially soluble in one of the domain types but not in theother(s), then one can test for continuity of those domains by measuringthe transport of that gas through the sample. If this is possible, thentransport through the continuous domains (type B) in the micellar phaseshould be only slightly slower than that in the pure B polymer, whereasgas transport for a gas confined to A domains should be very low.

The shear modulus of a block copolymer-based micellar phase isdetermined largely by that of the polymer block forming the continuousdomains, polymer B in our convention. Thus, for example, in a PS-PIdiblock which is 10% PS, so that PS micelles form in a continuous PImatrix, the shear modulus would be close to that of pure polyisoprenewith only a slight increase due to the presence of the PS micelles.Interestingly, in the reverse case, with 90% PS and thus PI micelles ina continuous PS matrix, the elastomeric PI micelles can provide ashock-absorbing component which can improve the fracture characteristicsover those of pure, glassy polystyrene.

L2 phase: This phase is the same as the L1 phase except that the rolesof the polar region and the apolar region are reversed: the curvature ofthe polar-apolar interface is toward the polar domains, the interior ofthe micelles (if they exist) is water and/or other polar moieties, andthe apolar domains (typically alkane chains of a lipid) form acontinuous matrix—although it is possible for the polar domains also toconnect up to form a bicontinuous L2 phase. As above, this phase can beeither nanostructured or structureless.

Identification of the nanostructured L2 phase. The guidelines for makinga phase identification of the nanostructured L2 phase are the same asthose given above for the L1 phase, with the following modifications. Weneed only discuss the surfactant-based L2 phase, since in the blockcopolymer-based systems the two types of micellar phases (A in B, and Bin A) are equivalent, and above we discussed the identification of themicellar phase in block copolymer systems.

First, L2 phases are generally more prominent when the HLB is low, forexample with ethoxylated alcohol surfactants having a small number ofethylene oxide groups (usually 5 or less, with typical alkyl chainlengths), or with double-chained surfactants. In terms of phasebehavior, they generally occur at higher surfactant concentrations thaneven the reversed liquid crystalline phases: a location that is verycommon is for the L2 phase to border the reversed hexagonal phase athigher surfactant concentrations. For L1 phases which are notbicontinuous, it is the water self-diffusion which is very low, andmeasurement of the diffusion coefficiently (by pulsed-gradient NMR, forexample) should give a number on the order of 10⁻¹¹ m²/sec or less.Also, a Hosemann plot will give the size of the reversed micelles, whichwill essentially be the water domain size.

Microemulsion: A microemulsion may be defined as a thermodynamicallystable, low viscosity, optically isotropic liquid phase containing oil(apolar liquid), water (polar liquid), and surfactant. See alsoDanielsson, I. and Lindman, B. (1981) Colloids and Surfaces, 3:391.Thermodynamically stable liquid mixtures of surfactant, water and oilare usually referred to as microemulsions. While being macroscopicallyhomogeneous, they are structured on a microscopic length scale (10–1,000Angstrom) into aqueous and oleic microdomains separated by asurfactant-rich film. See Skurtveit, R. and Olsson, U. (1991) J. Phys.Chem. 95:5353. A key defining feature of a microemulsion is that itcontains an “oil” (apolar solvent or liquid), in addition to water andsurfactant; it is always microstructured by definition. In general,because of the strong tendency for oil and water to phase segregate, inthe absence of an organic solvent capable of co-solubilizing oil andwater (such as ethanol, THF, dioxane, DMF, acetonitrile,dimethylsulfoxide, and a few others), a clear, single-phase liquidcontaining water and surfactant must be a microemulsion, and one cansafely conclude on that basis alone that the phase is nanostructured.Note that a microemulsion can also be an L1 or L2 phase especially, ifit contains well-defined micelles; however, if it is an L1 phase thenthe micelles are necessarily swollen with oil. The microemulsion is ananostructured liquid phase. If a liquid with “oil,” water andsurfactant has a characteristic domain size larger than the nanometerrange, that is, in the micron range, then it is no longer amicroemulsion but rather a “miniemulsion” or plain emulsion; both of thelatter are non-equilibrium. The term microemulsion was introduced,despite the fact that L1 and L2 phases can contain oil, and can even bebicontinuous, because it is fairly common for three-componentoil-water-surfactant/lipid systems to evolve continuously fromwater-continuous to bicontinuous to oil-continuous with no phaseboundaries in between. In this case, it does not make sense to try toset a dividing point between the “L1” and “L2” regions of the phasediagram; so instead, one just refers to the whole region as“microemulsion”—recognizing that at the high-water-content end of thisregion the structure is that of an oil-swollen L1 phase, and at thehigh-oil-content end of this region the structure is that of an L2phase. (In terms of Venn diagrams, there are overlaps between themicroemulsions and L1 and L2 phases, though not between L1 and L2phases). As discussed below, the microstructure of microemulsions isquite generally describable in terms of a monolayer film of surfactantthat divides oil-rich domains from water-rich domains. Thissurfactant/lipid-rich dividing film can enclose to form micelles, orconnect up into a network structure to form a bicontinuousmicroemulsion.

It must be pointed out that an emulsion is not a nanostructured liquid,as the term is applied herein. To begin with, the characteristic lengthscale in an emulsion, which essentially is the average size of anemulsion droplet, is generally much larger than the characteristiclength scale in a nanostructured liquid, and falls in the range ofmicrons instead of nanometers. While recent efforts to produce emulsionswith submicron droplet sizes have given rise to smaller dropletemulsions and to the advent of the term “miniemulsion”, there remaincrucial differences which exclude emulsions and miniemulsions from therealm of nanostructured liquid phases as applied herein. Thenanostructured liquid phases described herein, including microemulsions,exist at thermodynamic equilibrium, in contrast to emulsions which arenot equilibrium phases but only metastable materials. Furthermore, ananostructured liquid which is acquiescent and fully equilibrated isoptically transparent, whereas an emulsion is generally opaque—ordinarymilk is an emulsion, for example. In addition, if one takes the model ofFriberg for the structure of an ordinary emulsion to be true, and thisis generally recognized in the field, then the distinction at themolecular scale can be seen to be dramatic. According to that model,emulsion droplets can generally be seen to be stabilized by interfacialfilms which upon microscopic examination typically prove to be films ofnanostructured liquid crystalline phase material; thus, these emulsionshave a hierarchical structure in which a nanostructured phase plays therole of a stabilizing layer between the main building blocks, which arethe emulsion droplets and the continuous medium. Our use of the term“nanostructured” instead of “microstructured” is based on the moreprecise and restricted nature of the term “nanostructured” and itsexclusion of other liquid phases which fall into an entirely differentrealm, such as emulsions. Clearly, simple geometric considerationsdictate that an emulsion which has droplets on the order of 10 micronsin size, and a stabilizing film which may be a liquid crystalline layer,is not appropriate as the interior of a microparticle of the presentinvention which generally has a size on the order of 1 micron.

Determination of nanostructured microemulsions. The methods andguidelines discussed above for determination of nanostructured L1 phasescarry over to the determination of nanostructured microemulsion phases,with the following variations.

For microemulsions which do not clearly fall under either the L1 phaseor the L2 phase descriptions—which is the remaining case to be treatedhere—we take note that many, if not most, of these are bicontinuous, andin the context of a single liquid phase containing oil, water andsurfactant, bicontinuity provides strong proof that the phase isnanostructured, since emulsions and other common liquids are neverbicontinuous. This issue has been addressed in “On the demonstration ofbicontinuous structures in microemulsions.” Lindman, B., Shinoda, K.,Olsson, U., Anderson, D. M., Karlstrom, G. and Wennerstrom, H. (1989)Colloids and Surfaces 38:205. The time-tested way to demonstratebicontinuity is to use pulsed-gradient NMR and measure the effectiveself-diffusion coefficients of both oil and water separately; generallyit is best to measure also the self-diffusion of the surfactant.Electrical conductivity can also be used to establish water continuity,although this is prone to problems associated with “hopping” processes.Fluoresence quenching has also been used for continuity determination.Sanchez-Rubio, M., Santos-Vidals, L. M., Rushforth, D. S. and Puig, J.E. (1985) J. Phys. Chem. 89:411. Small-angle neutron and x-rayscattering analyses have been used to examine bicontinuity. Auvray, L.,Cotton, L. R., Ober, R. and Taupin, J. (1984) J. Phys. Chem. 88:4586.Porod analysis of SAXS curves has been used to deduce the presence ofinterfaces, thus proving that a nanostructure is present. Martino, A.and Kaler, E. W. (1990) J. Phys. Chem. 94:1627. Freeze-fracture electronmicroscopy, with extremely fast rates of freezing, has been used tostudy microemulsions and is the result of decades of development onfixation methods for nanostructured liquids: a critical reviewdiscussing the methods and the reliability of the results has beengiven. Talmon, Y. in K. L. Mirtal and P. Bothorel (Eds), Vol. 6. PlenumPress, New York, 1986, p. 1581.

In the event that an oil-water-surfactant liquid phase is not clearly anL1 or L2 phase, and does not show strong evidence of bicontinuity, thenthe analysis to demonstrate that it is nanostructured can be fairlyinvolved and no single technique will suffice. In general, one wouldapply the measurements discussed in this section, such as SANS or SAXS,NMR self-diffusion, cryo EM, etc., to attempt to rationalize the datawithin the context of a model nanostructure.

L3 phase: L2-phase regions in phase diagrams sometimes exhibit “tongues”sticking out of them: long, thin protrusions unlike the normalappearance of a simple L2 phase region. This sometimes appears also withsome L1 regions, as described below. When one examines these closely,especially with X-ray and neutron scattering, they differ in afundamental way from L2 phases. In an L2 phase, the surfactant film isgenerally in the form of a monolayer with oil (apolar solvent) on oneside and water (polar solvent) on the other. By contrast, in this “L3phase” as these phases are called, the surfactant is in the form of abilayer with water (polar solvent) on both sides. The L3 phase isgenerally considered to be bicontinuous and, in fact, it shares anotherproperty with cubic phases: there are two distinct aqueous networksinterwoven but separated by the bilayer. So, the L3 phase is really verysimilar to the cubic phase but lacking the long-range order of the cubicphase. L3 phases stemming from L2 phases and those stemming from L1phases are given different names. “L3 phase” is used for thoseassociated to L2 phases, and “L3* phase” for those associated to L1phases.

Determination of the nanostructured L3 phase. Determination of the L3phase in distinction to the other liquid phases discussed herein can bea sophisticated problem, requiring the combination of several analyses.The most important of these techniques are now discussed. In spite ofits optical isotropy when acquiescent and the fact that it is a liquid,the L3 phase can have the interesting property that it can exhibit flowbirefringence. Often this is associated with fairly high viscosity,viscosity that can be considerably higher than that observed in the L1and L2 phases, and comparable to or higher than that in the lamellarphase. These properties are of course a result of the continuous bilayerfilm, which places large constraints on the topology and the geometry ofthe nanostructure. Thus, shear can result in the cooperative deformation(and resulting alignment) of large portions of the bilayer film, incontrast with, for example, a micellar L1 phase where independentmicellar units can simply displace with shear, and in any case amonolayer is generally much more deformable under shear than a bilayer.Support for this interpretation comes from the fact that the viscosityof L3 phases is typically a linear function of the volume fraction ofsurfactant Snabre, P. and Porte, G. (1990) Europhys. Len. 13:641.

Sophisticated light, neutron, and x-ray scattering methodologies havebeen developed for determination of nanostructured L3 phases. Safinya,C. R., Roux, D., Smith, G. S., Sinha, S. K., Dimon, P., Clark, N. A. andBellocq. A. M. (1986) Phys. Rev. Lett. 57:2718; Roux, D. and Safinya, C.R. (1988) J. Phys. France 49:307; Nallet, F., Roux, D. and Prost. J.(1989) J. Phys. France 50:3147. The analysis of Roux, et al. in Roux,D., Cates, M. E., Olsson, U., Ball, R. C., Nallet, F. and Bellocq, A.M., Europhys. Lett. purportedly is able to determine that thenanostructure has two aqueous networks, separated by the surfactantbilayer, which gives rise to a certain symmetry due to the equivalenceof the two networks.

Fortunately, determination of the nanostructured nature of an L3 phasebased on phase behavior can be more secure than in the case of typicalL1, L2, or even microemulsion phases. This is first of all because theL3 phase is often obtained by addition of a small amount (a few percent)of oil or other compound to a lamellar or bicontinuous cubic phase, orsmall increase of temperature to these same phases. Since these liquidcrystalline phases are easy to demonstrate to be nanostructured (Braggpeaks in X-ray, in particular), one can be confident that the liquidphase is also nanostructured when it is so close in composition to aliquid crystalline phase. After all, it would be extremely unlikely thatthe addition of a few percent of oil to a nanostructured liquidcrystalline phase would convert the liquid crystal to a structurelessliquid. Indeed, pulsed-gradient NMR self-diffusion measurements in theAerosol OT—brine system show that the self-diffusion behavior in the L3phase extrapolates very clearly to those in the nearby reversedbicontinuous cubic phase. This same L3 phase has been the subject of acombined SANS, self-diffusion, and freeze-fracture-electron microscopystudy. Strey, R., Jahn, W., Skouri, M., Porte, G., Marisman, J. andOlsson, U. in “Structure and Dynamics of Supramolecular Aggregates—S. H.Chen, J. S. Huang and P. Tartaglia, Eds., Kluwer Academic Publishers,The Netherlands. Indeed, in SANS and SAXS scattering analysis of L3phases, a broad interference peak is often observed at wave vectors thatcorrespond to d-spacings that are the same order of magnitude as thosein bicontinuous cubic phases that are nearby in the phase diagram, andthe author has developed a model for L3 phase nanostructure which is anextrapolation of known structures for bicontinuous cubic phases.Anderson, D. M., Wennerstrom, H. and Olsson, U., (1989) J. Phys. Chem.93:4532.

The Nanostructured Liquid Crystalline Phases of Utility

As a component of the coated particle the nanostructured liquidcrystalline phase material may be

a. a nanostructured normal or reversed cubic phase material,

b. a nanostructured normal or reversed hexagonal phase material,

c. a nanostructured normal or reversed intermediate phase material or

d. a nanostructured lamellar phase material.

The nanostructured liquid crystalline phases are characterized by domainstructures composed of domains of at least a first type and a secondtype (and in some cases three or even more types of domains) having thefollowing properties:

a) the chemical moieties in the first type domains are incompatible withthose in the second type domains (and in general, each pair of differentdomain types are mutually incompatible) such that they do not mix underthe given conditions but rather remain as separate domains (for example,the first type domains could be composed substantially of polar moietiessuch as water and lipid head groups, while the second type domains couldbe composed substantially of apolar moieties such as hydrocarbon chains:or, first type domains could be polystyrene-rich, while second typedomains are polyisoprene-rich, and third type domains arepolyvinylpyrrolidone-rich);

b) the atomic ordering within each domain is liquid-like rather thansolid-like, lacking lattice-ordering of the atoms; (this would beevidenced by an absence of sharp Bragg peak-reflections in wide-anglex-ray diffraction);

c) the smallest dimension (e.g. thickness in the case of layers,diameter in the case of cylinders or spheres) of substantially alldomains is in the range of nanometers (viz., from about 1 to about 100nm); and

d) the organization of the domains conforms to a lattice, which may beone-, two-, or three-dimensional and which has a lattice parameter (orunit cell size) in the nanometer range (viz., from about 5 to about 200nm), the organization of domains thus conforms to one of the 230 spacegroups tabulated in the International Tables of Crystallography andwould be evidenced in a well-designed small-angle x-ray scattering(SAXS) measurement by the presence of sharp Bragg reflections withd-spacings of the lowest order reflections being in the range of 3–200nm.

In the discussion of the identification of these liquid crystallinephases using deuterium NMR or self-diffusion measurements, it is assumedthat the liquid crystal is not polymerized. In the cases where it ispolymerized, these measurements will be strongly affected by thepolymerization and may not conform to the same rules that apply forunpolymerized liquid crystals. In particular, the self-diffusioncoefficients of surfactants can be dramatically reduced, as was reportedby the present author in Strom, P. and Anderson, D. M. (1992) Langmuir8:691. NMR spectra for polymerized cubic phases were calculated forcertain conditions by the present author in Anderson, D. M. (1990)Supplement to J. de Phys. C7-1.

Lamellar phase: The lamellar phase is characterized by:

1. Small-angle x-ray shows peaks indexing as 1:2:3:4:5 . . . in wavenumber.

2. To the unaided eye, the phase is either transparent or exhibits mildor moderate turbidity.

3. In the polarizing optical microscope, the phase is birefringent, andthe well-known textures have been well described by Rosevear and byWinsor (e.g., Chem. Rev. 1968, p. 1). The three most pronounced texturesare the “Maltese crosses”, the “mosaic” pattern, and the “oily streaks”patterns. The Maltese cross is a superposition of two dark bands(interference fringes) roughly perpendicular to each other, over aroughly circular patch of light (birefringence), forming a distinctivepattern reminiscent of the WWI German military symbol. The variations onthis texture, as well as its source, is thoroughly described in J.Bellare, Ph.D. Thesis, Univ. of Minnesota, 1987. The “mosaic” texturecan be envisioned as the result of tightly packing together a densearray of deformed Maltese crosses, yielding dark and bright patchesrandomly quilted together. The “oily streaks” pattern is typically seenwhen the (low viscosity) lamellar phase flows between glass andcoverslip; in this pattern, long curved lines are seen, upon closeinspection under magnification 400×), to be composed of tiny striationswhich run roughly perpendicular to the line of the curve, as ties makeup a railroad track (to be contrasted with the hexagonal texturediscussion below). In some cases, particularly if the phase is massagedgently between glass and coverslip for a period of time, the lamellarphase will align with its optic axis parallel to the line of sight inthe microscope, resulting in a disappearance of the birefringence.

For lamellar phases in surfactant-water systems:

1. Viscosity is low enough so that the material flows (e.g. when a tubecontaining the phase is tipped upside down).

2. The self-diffusion rates of all components are high comparable totheir values in bulk—e.g., the effective self-diffusion coefficient ofwater in the lamellar phase is comparable to that in pure water. Sincethe surfactants that form liquid crystals are usually not liquid atambient temperatures, the reference point for the self-diffusioncoefficient of the surfactant is not clear-cut; and in fact, theeffective (measured) self-diffusion coefficient of the surfactant in thelamellar phase is often taken to be the reference point for interpretingmeasurements in other phases.

3. If the surfactant is deuterated in the head group, and the ²H NMRbandshape measured, one finds two spikes with the splitting between themtwice what it is in the hexagonal phase.

4. In terms of phase behavior, the lamellar phase generally occurs athigh surfactant concentrations in single-tailed surfactant/watersystems, typically above 70% surfactant: in double-tailed surfactants,it often occurs at lower concentrations, often extending well below 50%.It generally extends to considerably higher temperatures than do anyother liquid crystalline phases that happen to occur in the phasediagram.

For lamellar phases in single-component block copolymer systems:

1. Shear modulus is generally lower than other liquid crystalline phasesin the same system.

2. In terms of phase behavior, the lamellar phase generally occurs atvolume fractions of the two blocks is roughly 50:50.

Normal hexagonal phase: The normal hexagonal phase is characterized by:

1. Small-angle x-ray shows peaks indexing as 1:✓3:2:✓7:3 . . . ingeneral, ✓(h²+hk−k²), where h and k are integers—the Miller indices ofthe two-dimensional symmetry group.

2. To the unaided eye, the phase generally transparent when fullyequilibrated, and thus often considerably clearer than any nearbylamellar phase.

3. In the polarizing optical microscope, the phase is birefringent, andthe well-known textures have been well described by Rosevear and byWinsor (e.g., Chem. Rev. 1968. p. 1). The most distinctive of these isthe “fan-like” texture. This texture appears to be made up of patches ofbirefringence, where within a given patch fine striations fan out givingan appearance reminiscent of an oriental fan. Fan directions in adjacentpatches are randomly oriented with respect to each other. A keydifference distinguishing between lamellar and hexagonal patterns isthat the striations in the hexagonal phase do not, upon closeexamination at high magnification, prove to be composed of finerstriations running perpendicular to the direction of the largerstriation, as they do in the lamellar phase.

For normal hexagonal phases in surfactant-water systems:

1. Viscosity is moderate, more viscous than the lamellar phase but farless viscous than typical cubic phases (which have viscosities in themillions of centipoise).

2. The self-diffusion coefficient of the surfactant is slow compared tothat in the lamellar phase: that of water is comparable to that in bulkwater.

3. The ²H NMR bandshape using deuterated surfactant shows a splitting,which is one-half the splitting observed for the lamellar phase.

4. In terms of phase behavior, the normal hexagonal phase generallyoccurs at moderate surfactant concentrations in single-tailed surfactantwater systems, typically on the order of 50% surfactant. Usually thenormal hexagonal phase region is adjacent to the micellar (L1) phaseregion, although non-bicontinuous cubic phases can sometimes occur inbetween. In double-tailed surfactants, it generally does not occur atall in the binary surfactant-water system.

For hexagonal phases in single-component block copolymer systems, theterms “normal” and “reversed” do not generally apply (although in thecase where one block is polar and the other apolar, these qualifierscould be applied in principle). The shear modulus in such a hexagonalphase is generally higher than a lamellar phase and lower a bicontinuouscubic phase, in the same system. In terms of phase behavior, thehexagonal phases generally occurs at volume fractions of the two blockson the order of 35:65. Typically, two hexagonal phases will straddle thelamellar phase with, in each case, the minority component being insidethe cylinders (this description replacing the ‘normal/reversed’nomenclature of surfactant systems).

Reversed hexagonal phase: In surfactant-water systems, theidentification of the reversed hexagonal phase differs from the aboveidentification of the normal hexagonal phase in only two respects:

1. The viscosity of the reversed hexagonal phase is generally quitehigh, higher than a typical normal hexagonal phase, and approaching thatof a reversed cubic phase. And,

2. In terms of phase behavior, the reversed hexagonal phase generallyoccurs at high surfactant concentrations in double-tailedsurfactant/water systems, often extending to, or close to, 100%surfactant. Usually the reversed hexagonal phase region is adjacent tothe lamellar phase region which occurs at lower surfactantconcentration, although bicontinuous reversed cubic phases often occurin between. The reversed hexagonal phase does not appear, somewhatsurprisingly, in a number of binary systems with single-tailedsurfactants, such as those of many monoglycerides (include glycerolmonooleate), and a number of nonionic PEG-based surfactants with lowHLB.

As stated above in the discussion of normal hexagonal phases, thedistinction between normal and ‘reversed’ hexagonal phases makes senseonly in surfactant systems, and generally not in single-component blockcopolymer hexagonal phases.

Normal bicontinuous cubic phase: The normal bicontinuous cubic phase ischaracterized by:

1. Small-angle x-ray shows peaks indexing to a three-dimensional spacegroup with a cubic aspect. The most commonly encountered space groups,along with their indexings are: 1a3d (#230), with indexing ✓6:✓8:✓14:4 .. . Pn3m (#224), with indexing ✓2:✓3:2:✓6:8: and 1m3m (#229), withindexing ✓2:✓4:✓6:✓8:✓10 . . .

2. To the unaided eye, the phase is generally transparent when fullyequilibrated, and thus often considerably clearer than any nearbylamellar phase.

3. In the polarizing optical microscope, the phase is non-birefringent,and therefore there are no optical textures.

For normal bicontinuous cubic phases in surfactant-water systems:

1. Viscosity is high, much more viscous than the lamellar phase and evenmore viscous than typical normal hexagonal phases. Most cubic phase haveviscosities in the millions of centipoise.

2. No splitting is observed in the NMR bandshape, only a single peak,corresponding to isotropic motion.

3. In terms of phase behavior, the normal bicontinuous cubic phasegenerally occurs at fairly high surfactant concentrations insingle-tailed surfactant/water systems typically on the order of 70%surfactant with ionic surfactants. Usually the normal bicontinuous cubicphase region is between lamellar and normal hexagonal phase regions,which along with its high viscosity and non-birefringence make itsdetermination fairly simple. In double-tailed surfactants, it generallydoes not occur at all in the binary surfactant-water system.

For bicontinuous cubic phases in single-component block copolymersystems, the terms “normal” and “reversed” do not generally apply(although in the case where one block is polar and the other apolar,these qualifiers could be applied in principle). The shear modulus insuch a bicontinuous cubic phase is generally much higher than a lamellarphase, and significantly than a hexagonal phase in the same system. Interms of phase behavior, the bicontinuous cubic phases generally occurat volume fractions of the two blocks on the order of 26:74. In somecases, two bicontinuous cubic phases will straddle the lamellar phasewith, in each case, the minority component being inside the cylinders(this description replacing the ‘normal/reversed’ nomenclature ofsurfactant systems), and hexagonal phases straddling thecubic-lamellar-cubic progression.

Reversed bicontinuous cubic phase. The reversed bicontinuous cubic phaseis characterized by:

In surfactant-water systems, the identification of the reversedbicontinuous cubic phase differs from the above identification of thenormal bicontinuous cubic phase in only one respect. In terms of phasebehavior, the reversed bicontinuous cubic phase is found between thelamellar phase and the reversed hexagonal phase, whereas the normal isfound between the lamellar and normal hexagonal phases: one musttherefore make reference to the discussion above for distinguishingnormal hexagonal from reversed hexagonal. A good rule is that if thecubic phase lies to higher water concentrations than the lamellar phase,then it is normal, whereas if it lies to higher surfactantconcentrations than the lamellar then it is reversed. The reversed cubicphase generally occurs at high surfactant concentrations indouble-tailed surfactant/water systems, although this is oftencomplicated by the fact that the reversed cubic phase may only be foundin the presence of added hydrophobe (“oil”) or amphiphile. The reversedbicontinuous cubic phase does not appear in a number of binary systemswith single-tailed surfactants such as those of many monoglycerides(include glycerol monooleate) and a number of nonionic PEG-basedsurfactants with low HLB.

It should also be noted that in reversed bicontinuous cubic phases,though not in normal, the space group #212 has been observed. This phaseis derived from that of space group #230. As stated above in thediscussion of normal bicontinuous cubic phases, the distinction between‘normal’ and ‘reversed’ bicontinuous cubic phases makes sense only insurfactant systems, and generally not in single-component blockcopolymer bicontinuous cubic phases.

Normal discrete (non-bicontinuous) cubic phase: The normalnon-bicontinuous cubic phase is characterized by:

1. Small-angle x-ray shows peaks indexing to a three-dimensional spacegroup with a cubic aspect. The most commonly encountered space group insurfactant systems is Pm3n (#223) with indexing ✓2:✓4:✓5: . . . . Insingle-component block copolymers, the commonly observed space group isIm 3m, corresponding to body-centered sphere-packings with indexing✓2:✓4:✓6:✓8: . . . .

2. To the unaided eye, the phase is generally transparent when fullyequilibrated, and thus often considerably clearer than any associatedlamellar phase.

3. In the polarizing optical microscope, the phase is non-birefringentand therefore there are no optical textures.

For normal discrete cubic phases in surfactant-water systems:

1. Viscosity is high, much more viscous than the lamellar phase and evenmore viscous than typical normal hexagonal phases. Most cubic phase haveviscosities in the millions of centipoise, whether discrete orbicontinuous.

2. Also in common with the bicontinuous cubic phases, there is nosplitting in the NMR bandshape, only a single isotropic peak.

3. In terms of phase behavior, the normal discrete cubic phase generallyoccurs at fairly low surfactant concentrations in single-tailedsurfactant water systems, typically on the order of 40% surfactant withionic surfactants. Usually the normal discrete cubic phase region isbetween normal micellar and normal hexagonal phase regions, which alongwith its high viscosity and non-birefringence make its determinationfairly simple. In double-tailed surfactants, it generally does not occurat all in the binary surfactant-water system. For discrete cubic phasesin single-component block copolymer systems, the terms “normal” and“reversed” do not generally apply (although in the case where one blockis polar and the other apolar, these qualifiers could be applied inprinciple). The shear modulus in such a discrete cubic phase isgenerally dependent almost entirely on the shear modulus of the polymerthat forms the blocks in the continuous phase. In terms of phasebehavior, the discrete cubic phases generally occur at very low volumefractions of one or other of the two blocks, on the order of 20% orless.

Reversed discrete cubic phase: The reversed discrete cubic phase ischaracterized by:

In surfactant-water systems, the identification of the reversed discretecubic phase differs from the above identification of the normal discretecubic phase in three respects:

1. In terms of phase behavior, the reversed discrete cubic phase isfound between the lamellar phase and the reversed hexagonal phase,whereas the normal is found between the lamellar and normal hexagonalphases: one must therefore make reference to the discussion above fordistinguishing normal hexagonal from reversed hexagonal. A good rule isthat if the cubic phase lies to higher water concentrations than thelamellar phase, then it is normal, whereas if it lies to highersurfactant concentrations than the lamellar then it is reversed. Thereversed cubic phase generally occurs at high surfactant concentrationsin double-tailed surfactant/water systems, although this is oftencomplicated by the fact that the reversed cubic phase may only be foundin the presence of added hydrophobe (‘oil’) or amphiphile. The reverseddiscrete cubic phase does appear in a number of binary systems withsingle-tailed surfactants, such as those of many monoglycerides (includeglycerol monooleate), and a number of nonionic PEG-based surfactantswith low HLB.

2. The space group observed is usually Fd3m. #227.

3. The self-diffusion of the water is very low, while that of anyhydrophobe present is high; that of the surfactant is generally fairlyhigh, comparable to that in the lamellar phase. As stated above in thediscussion of normal discrete cubic phases, the distinction between‘normal’ and ‘reversed’ discrete cubic phases makes sense only insurfactant systems, and generally not in single-component blockcopolymer discrete cubic phases.

Intermediate phases: The intermediate phase is characterized by:

These phases occur quite rarely and when they are found they generallyoccupy very narrow regions in the phase diagram. Presently thestructures of many of these are unknown or under debate. Theintermediate phases can be classified as follows:

Normal int(1) phases occur at lower surfactant concentration than thenormal bicontinuous cubic phase, adjacent to the hexagonal phase.Viscosity is generally low or moderately low, no higher than that of thenormal hexagonal phase. The phase is birefringent, with texturestypically similar to those of the hexagonal phase. Self-diffusion of thecomponents is very similar to those in the hexagonal phase. Small anglex-ray shows a lower-symmetry space group than the cubic phases,typically monoclinic. Fairly sophisticated NMR bandshape and SAXSanalyses can be used to distinguish this phase from the normal hexagonalphase. See Henriksson, U., Blackmore, E. S., Tiddy, G. J. T. andSoderman, O. (1992) J. Phys. Chem. 96:3894. Typically bandshapesplittings will be intermediate between those of hexagonal and the zerosplitting of the isotropic phase, which provides good evidence of anintermediate phase.

Normal int(2) is found at higher concentrations than the normalbicontinuous cubic phase, adjacent to the lamellar phase. These bearclose resemblance, both in terms of property and probably also in termsof structure, to the normal bicontinuous cubic phases, except that theyare birefringent and show differences in NMR bandshape and SAXSanalyses. Optical textures are somewhat unusual, in some casesresembling lamellar textures and in some resembling hexagonal, but thesecan be considerably coarser than either of the more common phases. As inthe int(1) phases, the space group is of lower symmetry, typicallyrhombohedral or tetragonal, requiring two unit cell parameters forcharacterization and making SAXS analysis difficult. In general, if thesquares of the d-spacing ratios cannot be fit to a simple integralscheme, then an intermediate phase structure is suspect.

Reversed int(2) is found at lower concentrations than the reversedbicontinuous cubic phase, adjacent to the lamellar phase. These arebirefringent and show unusual in NMR bandshape and SAXS analyses. As inthe int(1) and int(2) phases, the space group is of lower symmetry,typically rhombohedral or tetragonal, requiring two unit cell parametersfor characterization and making SAXS analysis difficult. SAXS analysisdifficult, though the presence of Bragg peaks in the SAXS spectrum whichdo not index to a cubic or hexagonal lattice (which have only onelattice parameter) is, together with optical birefringence, indicationof an intermediate phase. Space groups which are likely for bicontinuousintermediate phases have been discussed in a publication by the presentauthor, D. M. Anderson, Supplement to J. Physique, Proceedings ofWorkshop on Geometry, and Interfaces, Aussois, France, September 1990,C7-1-C7-18.

At the time that the coated particle 10 is being formed and the exteriorcoating 20 is not yet formed, it is highly desirable that thenanostructured liquid phase material or the nanostructured liquidcrystalline phase material or the combination be one that is inequilibrium with water (polar solvent) or, more precisely, with a diluteaqueous solution. Once the coated particle 10 has its exterior coating20, the foregoing nanostructured material need not be one that is inequilibrium with water. The liquid phases that can be in equilibriumwith water are:

-   -   L2 phase (a.k.a. reversed micelles),    -   microemulsion, and    -   L3 phase (but not the L3* phase).        These supplement the liquid crystalline phases that can be in        equilibrium with water:    -   reversed cubic phase,    -   reversed hexagonal phase,    -   reversed intermediate phase, and    -   lamellar phase.        The phases that can be in equilibrium with water are preferred        from the point of view of making coated particles of the present        invention. Preferably, in using the process described herein to        disperse a given phase as the matrix, it is desirable that the        phase be insoluble in water, or whatever solvent the particles        are dispersed in. Furthermore, when the interior phase has the        additional property that it is in equilibrium with excess        aqueous solution during formation of the particles, then        concerns of phase transformation are minimized. Similarly when        the interior phase is in equilibrium with excess aqueous        solution under the conditions encountered when and after the        particle coating is released, then the concerns of phase changes        are likewise minimized and in some applications this may be        advantageous.

Whereas insolubility in water (external solvent, in general) ispreferred for the matrix at the instant of particle formation, andfrequently also at the time of application, there are applications wheresolubility in water at the time of application is advantageous, and thiscan be accomplished with the instant invention. For example, consider amatrix composed of 20% C12E5 (pentaethylene glycol dodecyl ether) inwater. At 75° C., this composition produces an L3 phase which is inequilibrium with excess water (dilute solution) and thus thiscomposition would be readily dispersible at 75° C. If the applicationtemperature were between 0 and 25° C., however, then this interiorcomposition would be soluble in water, and in fact the C12E5 acts as anordinary water-soluble surfactant at room temperature. This could beadvantageous if a non greasy, non-comedogenic—and even cleansing—finalproduct is desired after release of the particle coating.

The nanostructured liquid phase material may be formed from:

-   -   a. a polar solvent and a surfactant or    -   b. a polar solvent, a surfactant and an amphiphile or hydrophobe        or    -   c. a block copolymer or    -   d. a block copolymer and a solvent.

The nanostructured liquid crystalline phase material may be formed from:

-   -   a. a polar solvent and a surfactant.    -   b. a polar solvent, a surfactant and an amphiphile or        hydrophobe, or    -   c. a block copolymer or    -   d. a block copolymer and a solvent.

Above under the heading Chemical Criteria, criteria were discussed whichcould be used to select operative polar and apolar groups in order tomake an operative surfactant. Thus, suitable surfactants include thosecompounds which contain two chemical moieties, one being an operativepolar group chosen from those described in that discussion of polargroups, and the other being an operative apolar group chosen from thosedescribed in that discussion of apolar groups.

Surfactants of Utility.

Suitable surfactants or block copolymer components (or mixtures thereof)may

-   -   include: a. cationic surfactant    -   b. anionic surfactant    -   c. semipolar surfactant    -   d. zwitterionic surfactant        -   i. in particular, a phospholipid        -   ii. a lipid mixture containing phospholipids, designed to            match the physico-chemical characteristics of a biomembrane    -   e. monoglyceride    -   f. PEGylated surfactant    -   g. one of the above but with aromatic ring    -   h. block copolymer        -   i. with both blocks hydrophobic, but mutually immiscible        -   ii. with both blocks hydrophilic, but mutually immiscible,        -   iii. with one block hydrophilic and the other hydrophobic,            i.e., amphiphilic)    -   i. a mixture of two or more of the above.

Suitable lipids include phospholipids (such as phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, or sphingomyelin), orglycolipids (such as MGDG, diacylglucopyranosyl glycerols, and Lipid A).Other suitable lipids are phospholipids (including phosphatidylcholines,phosphatidylinositols, phosphatidylglycerols, phosphatidic acids,phosphatidylserines, phosphatidylethanolamines, etc.), sphingolipids(including sphingomyelins), glycolipids (such as galactolipids such asMGDG and DGDG, diacylglucopyranosyl glycerols, and Lipid A), salts ofcholic acids and related acids such as deoxycholic acid, glycocholicacid, taurocholic acid, etc., gentiobiosyls, isoprenoids, ceramides,plasmologens, cerebrosides (including sulphatides), gangliosides,cyclopentatriol lipids, dimethylaminopropane lipids, and lysolecithinsand other lysolipids which are derived from the above by removal of oneacyl chain.

Other suitable types of surfactants include anionic, cationic,zwittenionic, semipolar, PEGylated, amine oxide and aminolipids.Preferred surfactants are:

-   -   anionic—sodium oleate, sodium dodecyl sulfate, sodium        diethylhexyl sulfosuccinate, sodium dimethylhexyl        sulfosuccinate, sodium di-2-ethylacetate, sodium 2-ethylhexyl        sulfate, sodium undecane-3-sulfate, sodium        ethylphenylundecanoate, carboxylate soaps of the form 1C_(n),        where the chain length n is between 8 and 20 and I is a        monovalent counterion such as lithium, sodium, potassium,        rubidium, etc.,    -   cationic—dimethylammonium and trimethylammonium surfactants of        chain length from 8 to 20 and with chloride, bromide or sulfate        counterion, myristyl-gammapicolinium chloride and relatives with        alkyl chain lengths from 8 to 18, benzalkonium benzoate,        double-tailed quaternary ammonium surfactants with chain lengths        between 8 and 18 carbons and bromide, chloride or sulfate        counterions,    -   nonionic PEGylated surfactants of the form C_(n)E_(m) where the        alkane chain length n is from 6 to 20 carbons and the average        number of ethylene oxide groups m is from 2 to 80, ethoxylated        cholesterol;    -   zwitterionics and semipolars—N,N,N-trimethylaminodecanoimide,        amine oxide surfactants with alkyl chain length from 8 to 18        carbons: dodecyldimethylammoniopropane-1-sulfate,        dodecyldimethylammoniobutyrate, dodecyltrimethylene di(ammonium        chloride); decylmethylsulfonediimine;        dimethyleicosylammoniohexanoate and relatives of these        zwitterionics and semipolars with alkyl chain lengths from 8 to        20.

Preferred surfactants which are FDA-approved as injectables includebenzalkonium chloride, sodium deoxycholate, myristyl-gamma-picoliniumchloride, Poloxamer 188, polyoxyl castor oil and related PEGylatedcastor oil derivatives such as Cremophor EL, Arlatone G, sorbitanmonopalmitate, Pluronic 123, and sodium 2-ethylhexanoic acid. Otherlow-toxicity surfactants and lipids, which are of at least relativelylow solubility in water, that are preferred for the present inventionfor products intended for a number of routes of administration, include:acetylated monoglycerides, aluminum monostearate, ascorbyl palmitatefree acid and divalent salts, calcium stearoyl lactylate, ceteth-2,choleth, deoxycholic acid and divalent salts,dimethyldioctadecylammonium bentonite, docusate calcium, glycerylstearate, stearamidoethyl diethylamine, ammoniated glycyrrhizin, lanolinnonionic derivatives, lauric myristic diethanolamide, magnesiumstearate, methyl gluceth-120 dioleate, monoglyceride citrate,octoxynol-1, oleth-2, oleth-5, peg vegetable oil, peglicol-5-oleate,pegoxol 7 stearate, poloxamer 331, polyglyceryl-10 tetralinoleate,polyoxyethylene fatty acid esters, polyoxyl castor oil, polyoxyldistearate, polyoxyl glyceryl stearate, polyoxyl lanolin, polyoxyl-8stearate, polyoxyl 150 distearate, polyoxyl 2 stearate, polyoxyl 35castor oil, polyoxyl 8 stearate, polyoxyl 60 castor oil, polyoxyl 75lanolin, polysorbate 85, sodium stearoyl lactylate, sorbitansesquioleate, sorbitan trioleate, stear-o-wet c, stear-o-wet m,stearalkonium chloride, stearamidoethyl diethylamine (vaginal),steareth-2, steareth-10, stearic acid, stearyl citrate, sodium stearylfumarate or divalent salt, trideceth 10, trilaneth-4 phosphate, DetainePB, JBR-99 rhamnolipid (from Jeneil Biosurfactant), glycocholic acid andits salts, taurochenodeoxycholic acid (particularly combined withvitamin E), tocopheryl dimethylaminoacetate hydrochloride, tocopherylphosphonate, tocopheryl peg 1000 succinate, cytofectin gs,1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane, cholesterol linkedto lysinamide or ornithinamide, dimethyldioctadecyl ammonium bromide,1,2-dioleoyl-sn-3-ethylphosphocholine and other double-chained lipidswith a cationic charge carried by a phosphorus or arsenic atom,trimethyl aminoethane carbamoyl cholesterol iodide, lipoic acid,O,O′-ditetradecanoyl-N-(alpha-trimethyl ammonioacetyl) diethanolaminechloride (DC-6-14),N-[(1-(2,3-dioleyloxy)propyl)]-N-N-N-trimethylammonium chloride,N-methyl-4-(dioleyl)methylpyridinium chloride (saint-2), lipidicglycosides with amino alkyl pendent groups,1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide,bis[2-(11-phenoxyundecanoate)ethyl]-dimethylammonium bromide,N-hexadecyl-N-10-[O-(4-acetoxy)-phenylundecanoate]ethyl-dimethylammoniumbromide, bis[2-(11-butyloxyundecanoate)ethyl]dimethylammonium bromide,3-beta-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol, vaxfectin,cardiolipin, dodecyl-N,N-dimethylglycine, and lung surfactant (Exosurf,Survanta).

Suitable block copolymers are those composed of two or more mutuallyimmiscible blocks from the following classes of polymers: polydienes,polyallenes, polyacrylics and polymethacrylics (including polyacrylicacids, polymethacrylic acids, polyacrylates, polymethacrylates,polydisubstituted esters, polyacrylamides, polymethacrylamides, etc.),polyvinyl ethers, polyvinyl alcohols, polyacetals, polyvinyl ketones,polyvinylhalides, polyvinyl nitriles, polyvinyl esters, polystyrenes,polyphenylenes, polyoxides, polycarbonates, polyesters, polyanhydrides,polyurethanes, polysulfonates, polysiloxane, polysulfides, polysulfones,polyamides, polyhydrazides, polyureas, polycarbodiimides,polyphosphazenes, polysilanes, polysilazanes, polybenzoxazoles,polyoxadiazoles, polyoxadiazoiidines, polythiazoles, polybenzothiazoles,polypyromellitimides, polyquinoxalines, polybenzimidazoles,polypiperazines, cellulose derivatives, alginic acid and its salts,chitin, chitosan, glycogen, heparin, pectin, polyphosphorus nitrilechloride, polytri-n-butyl tin fluoride, polyphosphoryldimethylamide,poly-2,5-selenienylene, poly-4-n-butylpyridinium bromide,poly-2-N-methylpyridinium iodide, polyallylammonium chloride, andpolysodium-sulfonate-trimethylene oxyethylene. Preferred polymer blocksare polyethylene oxide, polypropylene oxide, polybutadiene,polyisoprene, polychlorobutadiene, polyacetylene, polyacrylic acid andits salts, polymethacrylic acid and its salts, polyitaconic acid and itssalts, polymethylacrylate, polyethylacrylate, polybutylacrylate,polymethylmethacrylate, polypropylmethacrylate, poly-N-vinyl carbazole,polyacrylamide, polyisopropylacrylamide, polymethacrylamide,polyacrylonitrile, polyvinyl acetate, polyvinyl acprylate, polystyrene,poly-alpha-methylstyrene, polystyrene sulfonic acid and its salts,polybromostyrene, polybutyleneoxide, polyacrolein, polydimethylsiloxane,polyvinyl pyridine, polyvinyl pyrrolidone, polyoxy-tetramethylene,polydimethylfulvene, polymethylphenylsiloxane, polycyclopentadienylenevinylene, polyalkylthiophene, polyalkyl-p-phenylene,polyethylene-altpropylene, polynorbomene,poly-5-((trimethylsiloxy)methyl)norbomene, polythiophenylene, heparin,pectin, chitin, chitosan, and alginic acid and its salts. Especiallypreferred block copolymers are polystyrene-b-butadiene,polystyrene-b-isoprene, polystyrene-b-styrenesulfonic acid,polyethyleneoxide-b-propyleneoxide, polystyrene-b-dimethylsiloxane,polyethyleneoxide-b-styrene,polynorborene-b-5-((trimethylsiloxy)methyl)norbornene,polyacetylene-b-5((trimethylsiloxy)methyl)norbornene,polyacetylene-b-norbornene, polyethyleneoxide-b-norbornene,polybutyleneoxide-b-ethyleneoxide, polyethyleneoxide-b-siloxane, and thetriblock copolymer polyisoprene-b-styrene-b-2-vinylpyridine.

Third Component: Hydrophobe or Non-surfactant Amphiphile.

This component can serve multiple functions in a matrix of the presentinvention, including modulation of phase behavior, tuning of poresize,solubilization of an active, modulation of release properties etc.Choices appropriate for this invention include:

-   -   a. alkane or alkene, other long-chain aliphatic compound    -   b. aromatic compound, such as toluene    -   c. long-chain alcohol    -   d. a glyceride (diglyceride or triglyceride)    -   e. an acylated sorbitan, such as a sorbitan triester (e.g.,        sorbitan trioleate), or sesquioleate, or mixture of sorbitans        with different numbers of acyl chains between 2 and 6    -   f. other hydrophobe or non-surfactant amphiphile or mixture with        one or more of the above,    -   g. none.

Suitable third components (hydrophobes or non-surfactant amphiphiles),include: n-alkane, where n is from 6 to 20, including branched,unsaturated, and substituted variants (alkenes, chloroalkanes, etc.),cholesterol and related compounds, terpenes, diterpenes, triterpenes,fatty alcohols, fatty acids, aromatics, cyclohexanes, bicyclics such asnaphthalenes and naphthol, quinolines and benzoquinolines, etc.,tricyclics such as carbazole, phenothiazine, etc., pigments,chlorophyll, sterols, triglycerides, sucrose fatty acid esters (such asOlestra™), natural oil extracts (such as clove oil, anise oil, cinnamonoil, coriander oil, eucalyptus oil, peppermint oil), wax, bilirubin,bromine, iodine, hydrophobic and amphiphilic proteins and polypeptides(including gramicidin, casein, receptor proteins, lipid-anchoredproteins, etc.), local anesthetics (such as butacaine, ecgonine,procaine, etc.), and low-molecular weight hydrophobic polymers (seelisting of polymers above). Especially preferred third components are:anise oil, clove oil, coriander oil, cinnamon oil, eucalyptus oil,peppermint oil, beeswax, benzoin, benzyl alcohol, benzyl benzoate,naphthol, capsaicin, cetearyl alcohol, cetyl alcohol, cinnamaldehyde,cocoa butter, coconut oil, cottonseed oil (hydrogenated), cyclohexane,cyclomethicone, dibutyl phthalate, dibutyl sebacate, diocryl phthalate,DIPAC, ethyl phthalate, ethyl vanillin, eugenol, fumaric acid, glyceryldistearate, menthol, methyl acrylate, methyl salicylate, myristylalcohol, oleic acid, oleyl alcohol, benzyl chloride, paraffin, peanutoil, piperonal, rapeseed oil, rosin, sesame oil, sorbitan fatty acidesters, squalane, squalene, stearic acid, triacetin, trimyristin,vanillin, and vitamin E.

Polar Solvent.

The polar solvent (or in the case of a block copolymer, the preferentialsolvent) can similarly serve multiple functions, including modulation ofphase behavior (indeed, making nanostructured phases possible at all, inmany surfactant systems), solubilization of the active, providing apolar environment for portions of the active molecule such as forexample the polar regions of a protein, etc. The choice of anon-volatile polar solvent like glycerol can be important in processessuch as spray-drying. The polar solvent may be:

-   -   a. water    -   b. glycerol    -   c. formamide, N-methyl formamide, or dimethylformamide    -   d. ethylene glycol or other polyhydric alcohol    -   e. ethylammonium nitrate    -   f. other non-aqueous polar solvents such as N-methyl sydnone,        N-methyl acetamide, pyridinium chloride, etc.:    -   g. a mixture of two or more of the above.

Desirable polar solvents are water, glycerol, ethylene glycol,formamide, N-methyl formamide, dimethylformamide, ethylammonium nitrate,and polyethylene glycol.

It can be advantageous in certain circumstances to use, as the interiormatrix, a composition that yields a nanostructured liquid or liquidcrystalline phase upon contact with water (or more rarely, other polarsolvent)—whether or not this dehydrated composition itself is ananostructured liquid or liquid crystalline phase. In particular, thiscontact with water or a water-containing mixture could be either duringa reconstitution step, or more preferably, during the application of theparticle, most preferably after the coating releases, and the de-coatedparticle contacts an aqueous solution such as blood, extracellularfluid, intracellular fluid, mucous, intestinal fluid, etc. There areseveral reasons why this may be advantageous: to protect hydrolyticallyunstable actives or excipients, to limit premature release ofwater-soluble actives; and as a natural result of a production processsuch as spray-drying or freeze-drying that can induce dehydration.Removal of most, or all, of the water from a nanostructured liquid orliquid crystalline phase will often yield another nanostructured liquidor liquid crystalline phase, but can sometimes yield a structurelesssolution, precipitate, or a mixture of these with one or morenanostructured liquid or liquid crystalline phases. In any case for manyapplications, it is the hydrated form that is important in theapplication of the particles, and thus if this hydrated form is ananostructured liquid or liquid crystalline phase, then the compositionof matter falls within the scope of the current invention.

Coatings.

As previously stated, the exterior coating 20 may be formed of anonlamellar material. The term “nonlamellar” as applied to crystalstructure herein should be taken in the following context. Lamellarcrystalline materials, which are distinct from lamellar liquidcrystalline phases, occur in organic compounds (typically polar lipids),organic compounds, and organometallics. Although these materials can betrue crystalline materials and can thus exhibit long rangethree-dimensional lattice ordering of the constituent atoms (ormolecules, in the case of an organic crystalline material) in space, theforces and interactions between atoms—which can include covalentbonding, ionic bonding, hydrogen bonding, steric interactions,hydrophobic interactions, dispersion forces, etc.—are much strongeramongst the constituent atoms or molecules than within the plane of alamella than across distinct lamellae. For example, in the case of thelayered structure of graphite, the atoms within a layer are covalentlybonded with each other into a two-dimensional network, whereas betweendistinct layers there is no bonding, only the weaker dispersion forcesand steric interactions. This absence of strong local interlamellarinteractions gives rise to a number of physicochemical properties whichmake them undesirable as coating materials in the present invention.

To begin with, the physical integrity of lamellar crystals is inherentlycompromised by the weak local interactions between layers. This isdramatically evidenced by the comparison between graphite (a layeredcrystalline form of carbon) with diamond (a crystalline form of carbonthat has three-dimensional bonding). Indeed, the fact that graphite isan important ingredient in certain lubricants, due to the ease withwhich the layers slide over each other, whereas diamond is an abrasive,illustrates the “liquid-like” (or “liquid crystal-like”) character oflayered crystalline structures in terms of their response to shear. Thissame inter-lamellar sliding effect is, in fact, the same effect thatgives rise to the much lower viscosity of the lamellar liquidcrystalline phase compared with that of other liquid crystalline phases,in particular compared with the very high viscoelasticity of thebicontinuous cubic phase. As a further indication of this liquid-likenature, the Moh's hardness of graphite is 1.0, whereas that of diamondis 10. The loss of integrity with shear in the case of graphite is seenin everyday life with “lead” pencils, which are graphite.

The detrimental effects associated with layered crystalline structurescan be seen in every day life even in situations where macroscopic shearis not involved. According to a widely-accepted model of the structureof emulsions advanced by Stig Friberg, as reviewed by Larsson, K. and S.Friberag, Eds. 1990, Food Emulsions 2″ Edition, Marcel Dekker, Inc. NY,lamellar liquid crystalline or, commonly, lamellar crystalline coatingsstabilize the oil droplets in an oil-and-water emulsion, and the waterdroplets in water-in-oil emulsion. In commonly encountered emulsionssuch as milk, ice cream, mayonnaise etc., the instabilities that arewellknown to the lay person—and in the field referred to as “breaking”of emulsions—are due in large part to the fluidity of these layeredcoating materials. Even in a quiescent emulsion, these layered coatingsundergo continual disruption, streaming and coalescence, and with timeany emulsion must ultimately succumb to the destabilizing effect ofthese disruptions.

And at yet another level, layered crystalline materials exhibit chemicalinstabilities of the type that would prevent their application ascoatings in embodiments of the current invention. Consider the case ofthe Werner complexes isomorphous to nickel dithiocyanatetetra(4methylpyridine) that form elathrate compounds with a host latticecontaining embedded guest molecules, in most cases yielding permanentpores upon removal of the guest. One such Werner complex was used as thecoating in a particle in Example 22, thus illustrating the use of thepresent invention in creating particles with coatings possessing fixed,controlled-size and highselectivity pores. According to J. Lipkowski,Inclusion Compounds 1, Academic Press, London (1984), p. 59, “Layeredstructures of Ni(NCS)₂(4-MePy)₄ are stable only in the presence of guestmolecules while the beta-phases preserve their porosity even in theabsence of guest molecules.” The three-dimensional, non-layeredstructure of the beta-phases is discussed in detail in the samepublication, such as: “. . . beta-phases . . . have a three-dimensionalsystem of cavities interconnected through channels of molecular size”.

Nonlamellar amorphous and semi-crystalline materials are materialscomprising non-crystalline domains (or lacking crystallinity altogether)in which strong atomic interactions exist in all three dimensions. Inthe amorphous trehalose that provides the coating in Example 40, forexample, the packing of these sugar molecules and the multiple hydrogenbonds that each individual molecule can participate in make this acompound that exhibits strong interactions in all three dimensions (andthe amorphous property rules out any lamellar-type structure). Similarlyamorphous PLGA has strong interactions between the carboxyl groupsacross neighboring polymer chains which, since the material is opticallyisotropic, are not limited to two dimensions. The release of a coatingin a PLGA-coated particle will be chosen to be based on its hydrolysisrate in the body, as is well-known in the art, and not by mechanicalshear or deformation as could occur in a particle coated with a lamellarcoating. Since most production protocols used in industrial orpharmaceutical practice involve shear, release upon the application, ofsuch shear rates to a lamellar-coated particle system could bedetrimental or disastrous in the context of such a process.

As is well-known in the art, in the case of polymers, polymersuniversally have amorphous domains: no polymer is ever 100% crystalline,and thus even high-crystallinity polymers are semi-crystalline andpossess a finite fraction of amorphous domains. Often this is in therange of about 1–50%. The glass transition temperature of theseamorphous domains can usually be detected by thermodynamic (e.g., DSC)techniques or rheometric measurements, though in certain veryhigh-crystallinity polymers (greater than about 98%), this may be adifficult undertaking. Nevertheless, even in these high-crystallinitycases the amorphous domains can play important roles: they can mitigatestructural problems associated with microcrystallite boundaries, thusconferring greater homogeneity and cohesiveness to microcrystallinepolymers; this in turn can have strong effects on rheological propertiesand behavior as diffusional barriers; according to the fringed micellemodel, an amorphous domain can provide a medium that allows for a singlechain to extend through several microcrystallites, yielding a physicalcrosslinking (analogous to the physical crosslinking that occurs inthermoplastic elastomers); and their presence may in fact allow forcrystallinity in high-MW polymers where the amorphous domains are thenecessary result of chain folding. Being amorphous, these domains arenon-lamellar regions in the polymer that are distinct from thecrystalline regions but nonetheless actually play crucial roles in thecrystallization of polymers and in determining their overall properties.

The exterior coating 20 can protect the internal core 10 and any activeagent(s) or component(s) disposed therein, for example, againstoxidation, hydrolysis, premature release, precipitation, shear, vacuum,enzymatic attack, degradation from other components of the preparation,and/or conditions external to the coated particles, for example, intheir preparation such as pH, ionic strength, or the presence ofbioactive impurities such as proteases or nucleases. Examples of each ofthese are:

-   -   oxidation: e.g. for antioxidants such as vitamin C, which are by        their very nature sensitive to oxidation, or unsaturated lipids;    -   hydrolysis: e.g., for a drug with a labile ester bond;    -   premature release: during storage;    -   precipitation: e.g., for a drug in the protonated        (hydrochloride) form that would deprotonate at the body pH and        thereby become insoluble;    -   shear: e.g., in cases where processing after encapsulation        endangers shear-sensitive compounds, such as proteins;    -   vacuum: e.g., in cases where processing involves vacuum-drying;    -   enzymatic attack: a peptide hormone, such as somatostatin, which        is normally quickly digested by enzymes in the body, can be held        active in circulation until reaching the site of release and        action;    -   degradation from other components: e.g., where even a slight        reactivity between an component disposed in the internal core        and an exterior one could, over a shelf-life of months or years,        pose a problem;    -   external pH: e.g., a drug in protonated form could be        encapsulated at low internal pH to ensure solubility, but        without requiring a low pH of the exterior liquid which would        otherwise upset the stomach,    -   external ionic strength: e.g., where a protein is encapsulated        to avoid salting-out and denaturation;    -   external impurities such as proteases, nucleases, etc.: e.g.,        when the exterior contains a bioreactor-derived product from        which removal of proteases might be prohibitively expensive.

Examples of suitable nonlamellar coating materials, namely, compoundswhich occur in nonlamellar form over useful temperature ranges, andwhich are in most cases of low toxicity and environmental impact are:ascorbic acid; ascorbic palmitate; aspartic acid; benzoin;beta-naphthol; bismuth subcarbonate; butylated hydroxytoluene;butylparaben; calcium acetate; calcium ascorbate; calcium carbonate;calcium chloride; calcium citrate; calcium hydroxide; calcium phosphate,dibasic; calcium phosphate, tribasic; calcium pyrophosphate; calciumsalicyiate; calcium silicate; calcium sulfate; carmine; cetearylalcohol; cetyl alcohol; cinnamaldehyde; citric acid; cysteinehydrochloride; dibutyl sebacate; esculin; ferric oxide; ferric citrate;ferrosoferric oxide; gentisic acid; glutamic acid; glycine; gold;histidine; hydrochlorothiazide; iodine; iron oxide; lauryl sulfate;leucine; magnesium; magnesium aluminum silicate; magnesium carbonate;magnesium hydroxide; magnesium oxide; magnesium silicate; magnesiumsulfate; magnesium trisilicate; maleic acid; malic acid; DL-methylsalicylate; methylparaben; monosodium glutamate; propyl gallate;propylparaben; silica; silicon; silicon dioxide; sodium aluminosilicate;sodium aminobenzoate; sodium benzoate; sodium bicarbonate; sodiumbisulfate; sodium bisulfite; sodium carbonate; sodium chloride; sodiumcitrate; sodium metabisulfite; sodium nitrate; sodium phosphate,dibasic; sodium propionate; sodium salicylate; sodium stannate; sodiumsuccinate; sodium sulfate; sodium sulfate; sodium thiosulfate; sodiumthiosulfate; succinic acid; talc; talc triturate; tartaric acid;tartaric acid; DL-tartrazine; tellurium; titanium dioxide; triacetin;triethyl citrate; trichloromonofluorethane; tromethamine and2-hydroxy-n-cyclopropylmethyl morphinan hydrochloride; zinc oxide.

Calcium phosphate coatings are of interest in biomedical andpharmaceutical applications, since calcium phosphates are a majorcomponent of bone, teeth, and other structural components. For example,in the treatment of osteoporosis, the release of the appropriatepharmaceutical compound could be triggered by physiological conditionsthat induce dissolution of bone (and thus of the particle coating).

Potassium nitrate coatings are of interest in agricultural applicationssince the coating also act as plant fertilizers.

Iodine, aspartic acid, benzoic acid, butylated hydroxytoluene, calciumedetate disodium, gentisic acid, histidine, propyl gallate and zincoxide can be particularly useful as coatings in potential pharmaceuticalapplications because they have relatively low water solubility(generally less than 5%) and are on the FDA list of approved inactiveingredients for injectable formulations.

Of particular interest as coating materials are clathrates. Examples ofsuch materials are as follows:

1. Clathrates and inclusion compounds (some of which retain permanentporosity upon removal of the guest molecules): Werner complexes of theform MX₂A₄ where M is a divalent cation (Fe, Co, Ni, Cu, Zn, Cd, Mn, Hg,Cr), X is an anionic ligand (NCS—, NCO—, CN—, N0₃, (Cl—, Br—, I—), and Ais an electrically neutral ligand-substituted pyridine,alpha-arylalkylamine or isoquinoline, examples of A include4-methylpyridine, 3,5-dimethylpyridine, 4-phenylpyridine, and4-vinylpyridine. A wide range of guest molecules can be included inthese complexes, examples being benzene, toluene, xylene,dichlorobenzene, nitrotoluene, methanol, chloromethane, argon, krypton,xenon, oxygen, nitrogen, carbon dioxide, carbon disulfide, etc.:

reversible oxygen-carrying chelates such asbis-salicyladehyde-ethylenediiminecobalt and other bis salicyladehydeiminecobalt derivatives, cobalt(II) dihistidine and related cobalt(II)amino acid complexes, iron(II) dimethylglyoxime and nickel(II)dimethylglyoxime; and

complexes of the form K₂Zn₃[Fe(CN)_(o)]₂xILO, where certain values ofthe variable x correspond to complexes which yield permanent pores uponremoval of the water.

2. Zeolites:

faujasite-type NaX zeolite;

faujasite-type NaY zeolite; and

VPI-5 zeolite.

Amorphous and semi-crystalline nonlamellar materials.

In some embodiments of the present invention, the exterior coating ofthe particles of the present invention comprises nonlamellar materialswhich are not entirely in crystalline form. Such non-crystallinematerials may be amorphous or semicrystalline. In the art, the term“amorphous” as applied to materials means lacking long-range order; thisis in direct contrast to the case of a crystalline material, in whichthere is long-range order in the positions of atoms, such that theirpositions conform to a lattice with its associated periodicity. Thex-ray diffraction pattern of an amorphous material will be absent of anyBragg reflections, and any short-range correlations can at most giverise to broad maxima in the diffraction pattern, maxima which exhibitneither the sharpness nor the functional form of a true Braggreflection. By “semi-crystalline” is meant a material which has amixture of crystalline domains and amorphous domains.

Those of skill in the art will recognize that many materials can existin a crystalline, an amorphous, or a semicrystalline form, depending onthe preparation of the material. For example, many materials whichotherwise occur in crystalline form instead occur in amorphous form whenspray-dried, freeze-dried (as exemplified in Example 40, below), orprepared in other methods that are of central importance in the food,cosmetic, and pharmaceutical industries.

Amorphous materials have a number of properties which make themadvantageous for certain embodiments of the current invention. Forexample, one property of amorphous materials is that they are generallyfaster-dissolving than a corresponding (or comparable) material incrystalline form, and this can be advantageous in cases where fastdissolution of the exterior coating is desirable. Further, amorphousmaterials can be superior to their corresponding crystalline forms incertain material properties. For example, amorphous materials tend toexhibit higher ductility, and thus allow the adsorption of stresswithout cracking.

In general, small-molecule amorphous materials tend to exhibit lesserstability over time than their corresponding crystalline materials. Inparticular, a small-molecule amorphous material will often show atendency to revert to a crystalline form over a period of time that iscomparable to, or shorter than, timescales that are relevant for thestorage and use of a product. In the case of high-MW polymers, eventhough the true equilibrium condition may be a crystal, kinetics ofrearrangement can be so slow that the timescale required for attainmentof this equilibrium is for all intents and purposes infinite, so thatthe material can be locked into an amorphous or semi-crystalline state.For certain applications, this may be highly desirable. For example,many of the well-known elastomers and plastics, such as natural rubber(an example of an elastomer) or polymethylmethacrylate (PMMA, also knownas Plexiglass, an example of a thermoplastic), are amorphous materials.

Semi-crystalline materials can in certain ways offer significantadvantages, though their occurrence as long-lasting states is largelylimited to high-MW polymers. A semi-crystalline polymer with highcrystallinity can offer high modulus due to the preponderance ofcrystalline domains, but a certain amount of ductility due to thepresence of amorphous domains, which can absorb stress without cracking.A number of the most important polymers, both commodity and engineeringplastics, are semi-crystalline.

Examples of materials which occur in an amorphous or semi-crystallineform that may be utilized in the practice of the present inventioninclude: polydienes, polyallenes, polyacrylics and polymethacrylics(including polyacrylic acids, polymethacrylic acids, polyacrylates,polymethacrylates, polydisubstituted esters, polyacrylamides,polymethacrylamides, etc.), polyvinyl ethers, polyvinyl alcohols,polyacetals, polyvinyl ketones, polyvinylhalides, polyvinyl nitriles,polyvinyl esters, polystyrenes, polyphenylenes, polyoxides,polycarbonates, polyesters, polyanhydrides, polyurethanes,polysulfonates, polysiloxane, polysulfides, polysulfones, polyamides,polyhydrazides, polyureas, polycarbodiimides, polyphosphazenes,polysilanes, polysilazanes, polybenzoxazoles, polyoxadiazoles,polyoxadiazoiidines, polythiazoles, polybenzothiazoles,polypyromellitimides, polyquinoxalines, polybenzimidazoles,polypiperazines, cellulose derivatives, alginic acid and its salts, gumarabic and its salts, gelatin, PVP, tragacanth, agar, agarose, guar gum,carboxymethylcellulose, arabinogalactan. Carbopol, chitin, chitosan,Eudragits, glycogen, heparin, pectin, sugars (such as trehalose,lactose, maltose, and sucrose, or mixtures of sugars with albumin) andmore complex carbohydrates, as well as amorphous forms of the coatingmaterials listed above in connection with crystalline coating materials,obtained by processes that hinder crystallization, such as spray-drying,vitrification, etc.

In any case, taking the larger view, the availability of the fullspectral range of amorphous, semi-crystalline and crystalline materialsyields great power and flexibility to the technology of creatingparticles with nanostructured liquid and liquid crystalline interiors.The case of lactide-glycolide copolymers provides a particularlypertinent example, because these copolymers are amorphous over a rangeof lactide:glycolide ratios, and crystalline over other ranges. Byadjusting this ratio, it is possible to alter the form of the materialand thus its properties, thereby “tuning” the rate of hydrolysis of thecoating material. This, in turn, “tunes” the rate of release of activeagents disposed in either the coating or the particle interior.

Proteins, and perhaps to a lessor extent polypeptides, can also provideamorphous and semi-crystalline coating materials with advantageousproperties. Due to the intimacy of interactions that are well-knownbetween proteins and lipid matrices, the crystallization of a protein inan aqueous dispersion of nanostructured liquid or liquid crystallineparticles, preferably of the reversed bicontinuous cubic phase, couldyield particles of the instant invention wherein the coating wascomposed of semi-crystalline protein. Alternatively, gelation orprecipitation of a protein at the surface of a nanostructured liquid orliquid crystalline particle could yield a particle of the instantinvention wherein the coating was composed of amorphous protein. Thepresence of protein in the coating of such particles could serve one ormore important roles, including: targeting (that is, the coating itselfcould serve a dual role as a targeting compound); inhibition ofunfavorable protein adsorption (e.g., albumin binding; presentation of abiocompatible particle surface that would minimize uptake by the body'sdefenses (e.g., the RES) and yield long circulation times; andfunctional proteins that could perform metabolic functions at the siteof delivery that might yield enhanced absorption, diminished drugdegradation/metabolism, and/or regulation of cellular processes inconcert with the drug action. Furthermore, since the release of thecoating could be in response to enzymatic degradation (by e.g.,proteases), this could provide a means by which to achieve slow release,or targeted release to sites of accelerated metabolism.

Applications of the Invention

The coated particles 1 of the present have application in a variety offields. The coated particles 1 are adapted to absorb one or morematerials from a selected environment, adsorb one or more materials froma selected environment or release one or more materials, such as activeagents, disposed in the matrix. With respect to absorption, the coatedparticles may be used to harvest products or scavenge waste, inbiological or chemical reaction processes, to carry catalysts in thoseprocesses, to remove toxins, antigens or waste products in medicalapplications, to identify a few examples.

With respect to adsorption, the coated particles may be used aschromatographic media and as adsorbents.

With respect to release, the coated particles may be used for thecontrolled release of pharmaceutical agents such as anticancer agents orphotodynamic therapy agents, or cosmetic or cosmeceutical materials. Anactive agent may be disposed in the matrix for release upon thetriggering of release. For example, a pharmaceutical or biologicallyactive material may be disposed in the matrix, that is, it may be eitherdissolved, or dispersed, or in some cases be partially dissolved and theremainder dispersed.

In applications of these microparticles in drug-delivery or withembedded proteins or polypeptides (in particular receptor proteins), itcan be highly advantageous to have an interior matrix which, althoughsynthetic or semisynthetic, is designed to simulate closely thephysiochemical properties of a natural biomembrane from a living cell.This could be important for the proper functioning of a receptor proteinor other membrane component, for example, or for promoting assimilationof the interior matrix into the natural biomembrane in drug delivery, orespecially in targeting of the microparticles. Physiochemical propertiesthat can be important in such a context include the bilayer rigidity, (ameasure of the resistance to bending), bilayer fluidity (a measure ofthe microviscosity of the bilayer interior), the acyl chain length andbilayer thickness, the order parameter as a function of position on thelipid acyl chains, the surface charge density, the presence or absenceof segregated lipid domains of differing composition within the bilayer,bilayer curvature and monolayer curvature (for a discussion of therelationship between these two curvatures see H. Wennerstrom and D. M.Anderson, in Statistical Thermodynamics and Differential Geometry ofMicrostructured Materials, Eds. H. T. Davis and J. C. C. Nitsche,Springer-Verlag, 1992, p. 137), cholesterol content, carbohydratecontent, and the lipid protein ratio. By proper choice of composition,one can adjust these parameters to a large extent in an artificialsystem, namely a nanostructured liquid phase or liquid crystallinephase. For example, the bilayer rigidity can be reduced by the additionof amphiphiles, particularly aliphatic alcohols, and bilayer charge canbe adjusted by adjusting the ratio between uncharged lipids (such asphosphatidylcholine) and charged lipids (such as phosphatidic acid).Also, the addition of cholesterol is important for the function of anumber of membrane proteins. The lamellar phase, the reversedbicontinuous cubic phase, the L3 phase, and to a lesser extent thereversed hexagonal phase are in particular well suited for thisapproach. Thus, a particle of the present invention, with the interiormatrix being such a phase with tuned physiochemical characteristics forthe functioning of incorporated proteins or other biomolecules, can bevery valuable in products for pharmaceutics, clinical assays,biochemical research products, etc.

Membrane proteins are generally dependent on a bilayer milieu in orderto function properly and even to maintain proper conformation, and forsuch proteins the present invention—particularly with the bilayerproperties tuned as described above—could be an excellent and veryuseful matrix. Examples of membrane proteins include, in addition toreceptor proteins, such proteins as proteinase A, amyloglucosidase,enkephalinase, dipeptidyl peptidase IV, gamma-glutamyl transferase,galactosidase, neuraminidase, alpha-mannosidase, cholinesterase,arylamidase, surfactin, ferrochelatase, spiralin, penicillin-bindingproteins, microsomal glycotransferases, kinases, bacterial outermembrane proteins, and histocompatibility antigens.

In view of the demanding requirements for the delivery ofpharmaceuticals in the treatment of cancers, the advantages andflexibility of the present intention make it particularly attractive inthe delivery and release of antineoplastic, such as for example, thefollowing:

Alkylating Agents

-   Alkyl Sulfonates—Busulfan, Improsuflan, Piposulfan,-   Aziriaines—Benzodepa, Carboquone, Meturedepa, Uredepa,-   Ethyleneimines and Methylmelamines—Altretamine, Triethylenemelamine,    Triethylenephosphoramide, Triethylenethiophosphoramide,    Trimethylolmelamine,-   Nitrogen Mustards—Chlorambucil, Chloramphazine, Cyclophosphamide,    Estramustine, Ifosfamide, Mechlorethamine, Mechlorethamine Oxide    Hydrochloride, Melphalan, Novembichin, Phenesterine, Prednimustine,    Trofosfamide, Uracil, Mustard,-   Nitrosourea—Carmustine, Chlorozotocin, Fotemustine, Lomustine,    Nimustine, Ranimustine,-   Others—Dacarbazine, Mannomustine, Mitobronitol, Mitolactol,    Pipobroman,    Antibiotics—Actacinomveins—Actinomycin FI, Anthramycin, Azaserine,    Bleomvyins, Cactinomycin, Carubicin, Carzinophilin, Chromomycins,    Dactinomycin, Daunorubicin, 6-Diazo-5-OXO-Leucine, Doxorubicin,    Epirubicin, Mitomycins, Mycophenolic Acid, Nogalamycin, Olivomycins,    Peplomycin, Plicarmcin, Porfiromycin, Puromycin, Streptonigrin,    Streptozocin, Tubercidin, Ubenimex, Zinostatin, Zorubicin.    Antimetabolites-   Folic Acid Analogs—Denopterin, Methotrexate, Pteropterin,    Trimetrexate,-   PurineAnalogs—Fludarabine, 6-Mercaptopurine, Thiamiprine,    Thioguanine,-   Pyrimidine Analogs—Ancitabine, Azacitidine, 6-Azauridine, Carmofur,    Cytarabine, Doxifluridine, Enocitabine, Floxuridine, Fluorouracil,    Tegafur.-   Enzymes—L-Asparaginase, etc.-   Others—Aceglatone, Amsacrine, Bestrabucil, Bisantrene, Carboplatin,    Cisplatin, Defosfamide, Demecolcine, Diaziquone, Eflorithine,    Elliptinium Acetate, Etoglucid, Etoposide, Gallium Nitrate,    Hydroxyurea, Interferon-ot, Interferon-P, Interferon-y,    Interleukin-2, Lentinan, Lonidamine, Mitoguazone, Mitoxantrone,    Mopidamol, Nitracrine, Pentostatin, Phenamet, Pirarubicin,    Podophyllinic Acid, 2-Ethylhydrazide, Procarbazine, PSK09, Razoxane,    Sizofiran, Spirogermanium, Taxol, Teniposide, Tenuazonic Acid,    Triaziquone, 2,2′,2,1,1-Trichlorotriethylamine, Urethan,    Vinblastine, Vincristine, Vindesine,-   Androgens—Calusterone, Dromostanolone Propionate, Epitiostanol,    Mepitiostane, Testolactone,-   Antiadrenals—Aminoglutethimide, Mitotane, Trilostane,-   Andandrogens—Flutamide, Nilutamide,-   Antiestrogen—Tamoxifen, Toremifene,-   Estrogens—Fosfestrol, Hexestrol, Polyestradiol Phosphate,-   LH-RH Analogs—Buserelin, Goserelin, Leuprolide, Triptorelin,-   Progestogens—Chlormadinone Acetate, Medroxyprogesterone, Megestrol    Acetate, Melengestrol.-   Antineoplastic (Radiation Source) Americium, Cobalt. ¹³¹I-Ethiodized    Oil, Gold (Radioactive, Colloidal), Radium, Radon, Sodium Iodide    (Radioactive), Sodium Phosphate (Radioactive),    Antineoplastic Adjuncts-   Folic Acid Replenisher—Folinic Acid,    Uroprotective—Mesna.

Other pharmaceutical compounds that are particularly well-suited forencapsulation according to the instant invention, and suffer fromproblems or limitations in the currently-marketed formulations, include:Dacarbazine, Ifosfamide, Streptozocin, Thiotepa. Nandrolone decanoate,Fentanyl citrate, Testosterone, Albendazole, Esmolol, Bleomycin sulfate,Dactinomycin, Amikacin sulfate, Gentamicin, Netilmicin, Streptomycin,Tobramycin, Doxorubicin, Epirubicin, Idarubicin, Valrubicin, Bacitracin,Colistimethate, Oxybutinin, Antithrombin III Human, Heparin, Lepirudin,Adenosine phosphate, Amphotericin B, Enalaprilat, Cladribine,Cytarabine, Fludarabine phosphate, Gemcitabine, Pentostatin, Docetaxel,Paclitaxel, Vinblastine, Vincristine, Vinorelbine, Batimastat,Rituximab, Trastazumab, Abciximab, Eptifibatide, Tirofiban, Droperidol,Aurothioglucose, Capreomycin disulfide, Acyclovir, Cidofovir,Pentafuside, Saquinavir, Ganciclovir, Cromolyn, Aldesleukin, Denileukin,Edrophonium, Infliximab, Doxapram, SN-38 (Irinotecan), Topotecan, Hemin,Daunorubicin, Teniposide, Trimetrexate, Octreotride, Ganirelix acetate,Histrelin acetate, Somatropin, Epoetin, Filgrastim, Oprelvekin,Leuprolide, Basiliximab, Daclizumab, Glatiramer acetate, Interferons,Muromonab-CD3, Cyclosporin A, Milrinone lactate, Buprenorphine,Nalbuphine, Urofollitropin, Desmopressin, Carboplatin, Cisplatin,Mitoxantrone, Estradiol. Hydroxyprogesterone, L-Thyroxine, Etanercept,Neostigmine, Epoprostenol, Methoxamine, Versed, Bupivacaine, Heparin,Insulin, Anitisense compounds, Ibuprofen, Ketoprofen, Alendronate,Etidronate, Zoledronate, Ibandronate, Risedronate, and Pamidronate.These compounds represent the following classes of drug: Alkylatingagent, Anabolic steroid, Analgesic, Androgen, Anthelmintic,Antiadrenergic, Antibiotic, Antibiotic, aminoglycoside, Antibiotic,antineoplastic, Antibiotic, polypeptide, Antichloinergic, Anticoagulant,Anticonvulsant, Antifungal, Antihypertensive, Antimetabolite,Antimitotic, Antineoplastic, Antiplatelet, Antipsychotic, Anesthetic,Antirheumatic, Antituberculosal, Antiviral, Antiviral (HIV), Asthmaanti-inflammatory, Biological response modifier, Cholinergic musclestimulant, CNS stimulant, DNA topoisomerase inhibitor, Enzyme inhibitor,Epipodophyllotoxin, Folate antagonist, Gastric antisecretory, Genetherapy agents, Gonadotropin-releasing, Growth hormone, Hematopoietic,Hormone, Immunologic agent, Immunosuppressant, Inotropic agent, Localanesthetic, narcotic agonist/antagonist, Ovulation stimulant, Pituitaryhormone, Platinum complex, Sex hormone, Thyroid hormone, TNF inhibitor(arthritis), Urinary cholinergic, Vasodilator, and Vasopressor. We notethat the current invention is also very well suited for theincorporation of functional excipients, such as gum benzoin or essentialoils that improve absorption of poorly-absorbed drugs, in some cases byinhibiting drug efflux proteins. As discussed in more detail elsewhereherein, there are a number of sites within, and at the surface of theparticles, where actives, excipients, and functional excipients can belocalized within the context of this invention.

Other examples of uses of coated particles of the present inventioninclude:

-   1. Paints and inks. Including Microencapsulation of pigments;    Cationic charging of pigments (where pH-dependence can be    important); Fillers and texturizing agents for non-aqueous paints;-   2. Paper. Including Microcapsular opacifiers (also in paints);    Pressure-sensitive ink microcapsules for carbonless copying paper;-   3. Non-wovens. Including Additives that adhere to fibers throughout    processing;-   4. Agricultural. Including controlled release of pheromones (some of    which are otherwise volatile or environmentally unstable if not    encapsulated) for insect control; Controlled release of insect    chemosterilants and growth regulators (many of which are otherwise    environmentally unstable): Controlled release of other pesticides    (with temperature independence being important); Controlled release    of herbicides: Encapsulation of the plant growth regulators ethylene    and acetylene (that are otherwise volatile); Taste modifiers to    deter mammalian pests (e.g. capsaicin), Nutrient and fertilizer    release:-   5. Environment and forestry. Including Controlled release of aquatic    herbicides for weed control; Controlled release of other herbicides;    Controlled release of nutrients in agriculture; Soil treatment and    nutrient release; Encapsulation and release of chelating agents    (e.g., for heavy metal contaminants); Control of deposition and    environmental fate of actives (viz., through targeted release of    crystal coating and/or adhesive property of cubic phase):    Encapsulation of hygroscopic or other (e.g., urea and sodium    chloride) “seeding” agents for meteorological control;-   6. Vaccines. Including HIV gag, gag-pol transfection of cells as an    example; Adjuvants for the proper presentation of antigens or    antibodies;-   7. Nuclear medicine. Including Separation of two (otherwise    mutually-destructive) radionuclides into separate particles for    treatment of cancer;-   8. Cosmetics. Including Antioxidant, Antiaging skin cream:    Separation of two components of an antiacne medication; Suntan    lotions with encapsulated prostaglandins and vitamins; Encapsulation    of fat-soluble vitamins, oxidatively sensitive vitamins, vitamin    mixes; Encapsulation of volatile perfumes and other odorants;    Encapsulated volatile perfumes for scratch and sniff advertisements,    Encapsulation of volatile make-up removers or other cosmetics for    sheet formation; Encapsulated solvents for nail polish removers (or    the polish itself); Aerosol particles containing encapsulated hair    dye; Sanitary napkins containing encapsulated deodorant;-   9. Veterinary. Including Controlled release of volatile anti-flea    compounds; Encapsulated feed additives for ruminants; Encapsulation    of anti-microbial and insecticides in animal husbandry;-   10. Dental. Including Controlled-release dentifrice components,    particularly hydrolytically unstable anti-calculus compounds;    Delivery of oral anti-cancer compounds (photophyrin);-   11. Polymerization catalysts in one-pot (single-package) resin    systems;-   12. Household Products. Including controlled-release air fresheners,    perfumes; Controlled-release insect repellants; Laundry detergents    (e.g., encapsulated proteases); Other detergency applications;    Softeners; Fluorescent brighteners;-   13. Industrial. Including encapsulation of phosphine, ethylene    dibromide, etc. volatiles for fumigating stored products; Catalytic    particles; Activated charcoal microparticles for sorption and    purification,-   14. Polymer additives. Including polymer additives for protection of    wires, paper cartons etc. from rodents; Impact modifiers; Colorants    and opacifiers; Flame retardant and smoke suppressants; Stabilizers:    Optical brighteners; Limitations in current polymer-based    encapsulation of additives include low melting point (during    processing, polymer-polymer incompatibility, particle size    limitations, optical clarity, etc. Some polymer additives used for    lubrication of the polymer are based on waxes, which suffer from low    melting point, except for certain synthetic waxes which are    expensive:-   15. Food and beverage processing. Including Encapsulation of    (volatile) flavors, aromas, and oils (e.g., coconut, peppermint);    Encapsulation of vegetable fats in cattle feeds; Encapsulated    enzymes for fermentation and purification (e.g., diacetyl reductase    in beer brewing); Encapsulation as an alternative to blanching, for    improved lifetime of frozen foods; Microencapsulated tobacco    additives (flavorings); pH-triggered buffering agents; Removal of    impurities and decolorization using activated charcoal encapsulated    in a porous material;-   16. Photographics. Including Fine-grain film with dispersions of    submicron photoreactive particles; Faster Film due to optical    clarity (and thus higher transmission) and shorter diffusion times    of submicron dispersion; Microencapsulation of photoprocessing    agents;-   17. Explosives and propellants. Including both liquid and solid    propellants and explosives are used in encapsulated form; also,    water is used in encapsulated form as a temperature moderator in    solid propellants;-   18. Research. Including Microcapsule-packed columns in extractions    and separations; Biochemical assays, particularly, in pharmaceutical    research and screening;-   19. Diagnostics. Including encapsulated markers for angiography and    radiography and clinical assays involving milieu-sensitive proteins    and glycolipids; indeed, particles incorporating certain radiopaque    or optically dense materials could themselves be used for imaging,    and when coupled to targeting compounds as described herein could    target specific sites in the body and allow their visualization.

Desirable triggers for commencing the release of active agents, oralternatively commencing absorption, are:

I. Release is by dissolution or disruption of the coating

-   -   A. Intensive variable        -   1. pH        -   2. Ionic strength        -   3. Pressure        -   4. Temperature    -   B. Extensive variable or other        -   1. Dilution        -   2. Surfactant action        -   3. Enzymatic activity        -   4. Chemical reaction (non-enzymatic)        -   5. Complexation with target compound        -   6. Electric current        -   7. Irradiation        -   8. Time (i.e. slow dissolution)        -   9. Shear (critical shear rate effective)

II. Release or absorption is via pores in the coating, circumventing theneed for dissolution or disruption of the coating

-   -   1. Selective by pore size vs. compound size    -   2. Selective by pore wall polarity vs. compound polarity    -   3. Selective by pore wall ionicity vs. compound ionicity    -   4. Selective by pore shape vs. compound shape    -   5. Selective by virtue of the fact that some compounds or ions        form porous inclusion compounds with the coating, whereas others        do not (although this is generally a combination of the above 4        effects).        Methods for Making Particles of the Invention.

In a preferred embodiment, the coated particle may be made by

1. providing a volume of the matrix that includes at least one chemicalspecies having a moiety capable of forming a nonlamellar material uponreaction with a second moiety and

2. contacting the volume with a fluid containing at least one chemicalspecies having the second moiety under nonlamellar solidmaterial-forming conditions so as to react the first moiety with thesecond moiety and subdividing the volume into particles by theapplication of energy to the volume, or performing this subdivision intoparticles before, and/or after the chemical reaction.

Alternatively, the coated particle can be made by one of the followingprocesses:

-   -   providing a volume of the matrix that includes a material in        solution in it that is capable of forming a nonlamellar material        that is insoluble in the matrix and causing the aforesaid        material to become insoluble in the matrix and subdividing the        volume into particles by the application of energy to the        volume:    -   dispersing particles of said matrix into a fluid that includes        at least one chemical species having a moiety capable of forming        a nonlamellar material upon reaction or association with a        second moiety and adding to said dispersion at least one        chemical species having said second moiety to react said first        moiety with said second moiety;    -   dispersing particles of said matrix into a fluid that includes        at least one chemical species having a moiety capable of forming        a nonlamellar material upon reaction or association with a        second moiety, adding to said dispersion at least one chemical        species having said second moiety to react said first moiety        with said second moiety, and subdividing the resulting material        into particles by the application of energy to said material:    -   dispersing a volume of said matrix in a form of said nonlamellar        material selected from the group consisting of liquefied form,        solution, or fluid precursor, and solidifying said nonlamellar        material by a techniques selected from the group consisting of        cooling, evaporating a volatile solvent, or implementing a        chemical reaction;    -   dispersing or dissolving a volume of said matrix in a liquid        comprising said nonlamellar material in solution or dispersed        form and comprising also a volatile solvent, and spray-drying        said solution or dispersion; or    -   applying spray-drying, electrospinning, or other comparable        process to a solution or dispersion that contains the components        of both the matrix and the coating. Or, a combination of these        processes can be used.

In a general method, a volume of the matrix is loaded with a compound Acapable of forming a nonlamellar material on reaction with compound B,and a fluid (typically an aqueous solution, often referred to as the“upper solution”) containing a compound B is overlaid on this, and thecontact between compound A and compound B induces precipitation at theinterior/exterior interface, which coupled with the application ofenergy, such as sonication, causes particles coated with the nonlamellarmaterial to break off into the fluid. This method of the presentinvention is uniquely well-suited for producing aqueous dispersions ofcoated particles having coatings of materials with low watersolubilities, i.e., preferably less than about twenty (20) grams perliter of water and even more preferably less than about ten (10) gramsper liter of water. It is highly advantageous in these processes forcomponent A to be dissolved (not merely dispersed or suspended) in thematrix before the contact with B and sonication begins, in order toobtain a homogeneous dispersion of microparticles in the end. Asdiscussed above, this is one reason (in addition to requirements foroptimizing solubilization of actives, particularly biopharmaceuticals,in the matrix) for the importance of a nanostructured matrix havingaqueous microdomains, in order to allow for the solubilization ofcompound A which in many cases is soluble only in polar solvents. Inparticular, reactions yielding nonlamellar organic precipitates aregenerally performed most conveniently and effectively in aqueous media,and reactions yielding nonlamellar organic precipitates from solubilizedprecursors are often most conveniently and effectively selected to be pHinduced protonation or deprotonation reactions of soluble salt forms ofthe desired nonlamellar exterior coating material, where water (or anaqueous microdomain) is an obvious medium.

Alternatively, a cool temperature, or a crystallization promoter, orelectric current could be used to produce precipitation.

In addition to sonication, other standard emulsification methods couldbe used as energy inputs. These include microfluidization, valvehomogenization [Thornberg, E. and Lundh, G., 1978) J. Food Sci. 43:1553]and blade stirring, etc. Desirably, a water-soluble surfactant,preferably an amphiphilic block copolymer of several thousand Daltonmolecular weight, such as Pluronic F68, is added to the aqueous solutionin order to stabilize the coated particles against aggregation as theyform. If sonication is used to promote particle formation, thissurfactant also serves to enhance the effect of sonication.

Many of the nonlamellar material-coated particles described in theExamples where made by a process in which two or more reactants react toform a precipitate at the interface between the external solution andthe nanostructured liquid or liquid crystalline phase, and theprecipitate forms the exterior coating. Another method which bearsimportant similarities, as well as important differences, to this methodis a general method in which the material that is to form the coating,call it material A, is dissolved in the liquid phase material or liquidcrystalline phase material, with this dissolution being promoted by thechange of one or more conditions in the material, such as an increase intemperature (but it could be another chance such as decrease inpressure, addition of a volatile solvent, etc.). This change must bereversible, so that upon reversal of the condition—decrease of thetemperature, increase in pressure, evaporation of solvent, etc.—thesystem reverts to a two-phase mixture of a nanostructured liquid orliquid crystalline phase material and a nonlamellar material A. Energyinput is applied, sometimes before the nonlamellar material A has thetime to coarsen into large precipitates, where this may be through theapplication of ultrasound, or other emulsification methodology. Thiscauses the breaking off of particles coated with nonlamellar material A.

For the case where temperature is used, the solubility of compound A inthe nanostructured liquid phase material or nanostructured liquidcrystalline phase material must change with temperature, and the higherthe magnitude of the slope of the solubility versus temperature plot,the smaller the temperature increment needed to perform this process.For example, the solubility of potassium nitrate in water is a verystrong function of temperature. A fundamental difference between theprecipitation reaction process and this type of process is that in thistype of process, only one compound (A) is needed in addition to thenanostructured interior matrix. In the precipitation reaction method, atleast two compounds are needed, component A which is in thenanostructured phase, and component B which starts out in the exteriorphase (“upper solution”) which is overlaid on top of the nanostructuredphase. Component B in that case is often simply a suitably chosen acidicor basic component. This serves to point out a similarity between thetwo processes, in that the presence of component B in the exterior phasecan alternatively be thought of as a “condition” (in particular, pH inthe acid/base case) which causes the precipitation of A, that is, A maybe solubilized by the use of basic pH, and this is reversible by the useof acidic pH conditions, which are applied by the presence of theexterior phase. Probably the most important distinction between the twomethods is whether the change in conditions that causes theprecipitation of A occurs only when and where the exterior phase (“uppersolution”) contacts the nanostructured phase, as in the A/Bprecipitation reaction, or whether it is occurring simultaneouslythroughout the bulk of the nanostructured phase, as in thetemperature-induced precipitation.

It is also possible to use a process which is a combination of the A/Breaction process and the temperature process described above. Typicallyin such a scheme, the compound desired as the particle coating would beadded to the matrix in two chemical forms. The first would be thechemical form of the final coating, typically the free acid (free base)form of a compound, which would be soluble only at elevated temperatureand insoluble in the matrix at the temperature of particular formation.The second would be a precursor form, typically the salt form made byreacting the free acid with a base such as sodium hydroxide (or reactingthe free base form with an acid such as hydrochloric acid), where thisprecursor form would be soluble in the matrix even at the temperature ofparticle formation. For example, for the case of a benzoic acid particlecoating, both benzoic acid and sodium benzoate would be added to thematrix, where the matrix is such that is does not dissolve benzoic acidat ambient temperature, but does at a higher temperature. The uppersolution would contain the necessary component(s) to convert theprecursor form to the final coating form, such as hydrochloric acid inthe case of sodium benzoate. Upon heating (so that both forms aresubstantially dissolved), and then cooling, overlaying the uppersolution, and sonicating or otherwise adding energy to the system, theformation of coated particles would involve the two methods ofcooling—induced precipitation and reaction—mediated formation andprecipitation of coating. This could have advantages, in terms ofproviding two sources of coating material that could result in particlecoverage at an earlier stage than with either method separately, thusproviding added protection against particle fusion (and possibly leadingto a more uniform particle size distribution), and more efficientparticle formation with less energy input requirement, etc. Othermethods that may be used for making coated particles of the presentinvention are: A. Electrocrystallization, B. Seeding (withsupersaturated solution in matrix, seed in exterior phase), C. Promotion(with supersaturated solution in one phase, crystallization promoter inthe other phase), D. Inhibition removal (with supersaturated solution inone phase and seed in the other phase), or E. Time method (precipitategrows slowly from supersaturated solution in interior phase).

In order to form many of the desired exterior coatings, including nearlyall of the inorganic ones, one of the reactants (and usually both) willinevitably be soluble only in water or other polar solvent. Inparticular, most of the salts that are used in these precipitationreactions will dissolve only in highly polar solvents. At the same time,in order for a matrix material to be dispersible in water in accordancewith the present invention, it appears to be a highly desirable, if notan absolute, condition that the interior phase material not be ofsubstantial solubility in water, otherwise some or all of the materialwill dissolve in the upper solution rather than be dispersed in it.Therefore, in order to form these coatings, the matrix must satisfy twoconditions:

condition 1: it must contain aqueous (or other polar solvent) domains:and

condition 2: it must be of low solubility in water, i.e., sufficientlylow (or with sufficiently slow dissolution kinetics) that substantialdissolution of the phase does not occur during the process of particleproduction from the phase (typically 5 to 100 minutes to disperse theentire material into particles) since this would substantially reducethe yield efficiency and could thus diminish the overall attractivenessof the method.

These two conditions are working in nearly opposite directions, and veryfew systems can be found that will satisfy both. Nanostructured liquidphase materials and liquid crystalline phase materials of the reversedtype or the lamellar type, are several of these very few systems. Insome cases, it will be advantageous to incorporate into the uppersolution one or more of the components that are in the nanostructuredliquid or liquid crystalline phase, and at times in appreciable amounts.Indeed, there are instances when it may be advantageous to have ananostructured surfactant-rich liquid phase for the upper solution. Inparticular, this could occur when the matrix phase is not in equilibriumwith water (or a dilute aqueous solution) but is in equilibrium withanother liquid or liquid crystalline phase, such as a micellar phase oreven a low-viscosity lamellar phase. Thus, as the “fluid” referred to inthe general description of the process given above, one could use such aphase, or such a phase to which additional components, such as reactantB and/or amphiphilic block copolymer stabilizer have been added. In thiscase there might well be no complications by any incorporation of thisupper phase into the microparticles, should that occur, since the upperphase could be (and generally would be) chosen so as to be inequilibrium with the matrix phase (except possibly for exchange of theactive ingredient between the two materials, which would have someconsequences but these would often be relatively unimportant). Afterformation of the coated particles, which would originally be dispersedin this upper “solution”, through the use of filtration or dialysis thecontinuous outer phase could be changed from this to another medium,such as water, saline, butter, etc.

In other embodiments of the present invention, advantages can beobtained by using a precursor to the coating material that localizespreferentially the surface of particles of the nanostructured liquid orliquid crystalline matrix, and dispersing the nanostructured liquid orliquid crystalline phase—often with the aid of this surface-localizedprecursor—prior to converting this precursor to the actual coatingmaterial. This is especially preferred in the case where asurface-active precursor can be found, or when the precursor canotherwise be substantially localized near the surface of the dispersedparticles, by a favorable interaction with another component (ionicpairing, hydrogen bonding, etc.), or by a non-specific effect such asthe hydrophobic effect, or by selecting a precursor orprecursor-containing solution with the proper surface energy. When thiscan be achieved, as it is in Example 41 below, then the localization ofthe precursor at the particle surface can be maintained throughout itsconversion to the coating resulting in good intimacy between theparticle and coating and efficient use of the coating material. InExample 41, the sodium salt of N-acetyltryptophan, which is asurface-active compound (due to the hydrophobicity ofN-acetyltryptophan, augmented by the polarity of the ionized carboxylategroup at one end), is used to disperse a cubic phase into microparticleswith a particle surface that is rich in this precursor to the finalcoating material, which in this case is the zinc salt ofN-acetyltryptophan. This is a very general approach, for example sincethe most useful coating materials are of course of low solubility inwater, and thus each possesses at least one dominant hydrophobic group,but also has at least one polar group that allows it to have sufficientsolubility or interaction with water in some precursor state; this is infact tantamount to saying that it is an amphiphile, or even asurface-active compound, in this precursor state (or that such a statecan be found). Other approaches for localizing the precursor at theparticle surface include: ion-pairing the precursor to anoppositely-charged molecule that partitions strongly into the cubicphase; using a melted or solubilized form of the precursor such that thesurface energy of the melted precursor or precursor solution favors itslocalization in between the nanostructured phase and the exterior phasein which the nanostructured phase is dispersed; choosing a precursorthat has favorable interactions such as extensive hydrogen bonding withthe nanostructured phase surface, particularly in the case where theprecursor (and coating) is a polymer, so that it is by virtue of itshigh MW excluded from the interior of the nanostructured phase particle;invoking specific interactions such as antibody-antigen orreceptor-ligand interactions; and using a precursor, preferably apolymer or biomacromolecule (protein, nucleic acid, polysaccharide,etc.) that is substantially insoluble in the nanostructured phase butcontains hydrophobic anchor groups that partition into thenanostructured phase, where such hydrophobic anchors are known in theart and typically are alkanes or cholesterol-derivatives that aregrafted onto the polymer or biomacromolecule.

As exemplified in Example 42, a related approach is one in which thematrix is dispersed in the precursor itself. That is, the precursorforms the continuous (exterior) phase of a dispersion of microparticlesof the matrix. Then, this precursor is converted to the coatingmaterial, entrapping the microparticles (if and when they remain asnanostructured liquid or liquid crystalline bodies) within the coatingmaterial.

In this type of approach, there is first the step of dispersing thematrix material, with in many cases the precursor playing a central roleas a dispersant or matrix, followed by the step of converting theprecursor to the coating material, be it by chemical reaction (often assimple as an acid-base reaction or formation of a complex by theintroduction of multivalent ions, as in Example 41), cooling,evaporating a volatile solvent, or other method. This series of actionscan result either in a dispersion of coated microparticles, orconglomerates of such particles which one may want to separate by asecond dispersing step (or it can yield a combination of conglomeratesand dispersed microparticles). In both Examples 41 and 42, macroscopicparticles were the result of these two steps, and a second dispersingstep is required if the resulting contiguous solid is to be reduced tomicroparticles, as was performed in Example 41, where the final resultwas a dispersion of submicron-sized, coated microparticles.

As discussed above, in certain embodiments of this invention theinterior matrix will be a dehydrated variant of the desired phase thatwill form the desired nanostructured liquid or liquid crystalline phaseupon contact with a water-containing fluid. There are three general waysin which such a particle can be produced. One is to use a processsimilar to that used in Example 42, where a matrix or, in this casedehydrated matrix, is dispersed in non-aqueous solution or melt that is,or contains, a precursor of the coating material; upon cooling orotherwise converting, this precursor to the coating, the dehydratedmatrix would then be the encapsulated entity. A second general method isto apply a drying process, such as freeze-drying, electrospinning, orpreferably spray-drying, to a water-containing dispersion of theparticles in which the coating material (or a precursor thereof) hasbeen dissolved or very finely dispersed. And a third general method isto dissolve or disperse all the components of the coating and of thematrix, either including or excluding the water, in a volatile solventand applying a drying process, again preferably spray-drying. Several ofthese methods can avoid the use of water completely, which would beimportant in the case of actives (or special excipients) that should notcontact water even during production.

Incorporation of Targeting Groups and Bioactive Compounds.

The utilization of amorphous and semi-crystalline materials as exteriorcoatings in the instant invention makes it all the more practical toincorporate, in a number of different ways, chemicals or chemical groupsthat can be invoked to target particles temporally and spatially, forexample, to target particles to specific sites in the body. Similarly,other bioactive compounds incorporated on or in the coatings could serveimportant functions, such as: absorption enhancers such as menthol couldbe present so as to increase permeability of absorption barriers (lipidbilayers, gap junctions) prior to or concomitant with the release ofdrug; proteins or other adsorption-modulating materials could beincorporated that would inhibit unfavorable binding of endogenousproteins such as albumin; adjuvants could be incorporated that wouldenhance the effect of vaccine components or other immune modulatingmaterials. In general, an amorphous or semi-crystalline material can,for example, incorporate molecules or even submicron solids as embeddedmaterials, more readily and efficiently than with crystalline materialswhich tend to exclude other materials during theircrystallization—particularly when the crystallization is performed inaccordance with the tight regulations that govern the pharmaceuticalindustry. Furthermore, covalent or ionic attachment of organic groups topolymers at their surfaces is a well-developed art. U.S. Pat. Nos.6,344,050 and 5,484,584 (incorporated herein by reference in theirentirety) are examples of methods known in the art for attachingmolecular targets to polymers and microparticle coatings in particular.Antibodies, steroids, hormones, oligo- or polysaccharides, nucleicacids, vitamins, immunogens, and even nanoprobes are all examples of awide range of materials that could be attached to particles of theinstant invention with an exterior phase of amorphous, semi-crystalline,or less likely crystalline, material.

It is also within the scope of this invention to use pharmaceuticalactives themselves as coatings, with the nanostructured interior playingone or more of several roles: enhancing absorption by virtue ofsurfactancy and/or interactions with biomembrane; solubilizing and thenreleasing absorption enhancers (e.g., gum benzoin), acids, bases,buffers, specific ions (e.g., manganese in the case where lectin bindingis important), modulators of protein binding or activity, or otherbioactive materials; and providing a matrix ensuring the properpresentation of molecular recognition sites.

While it is not always crucial for a given application to know the exactlocalization (or more precisely, the spatial probability distribution)of a targeting moiety within or in association with a particle, this maybe an important consideration in the design of a particle-targetingmoiety combination, and the instant invention lends itself to a gooddeal of flexibility and power in this respect. Typically, targetingmoieties could be substantially localized at one or more of thefollowing sites in reference to the coated microparticle:

-   1) in the interior of the particle, i.e., dissolved or dispersed in    the nanostructured liquid or liquid crystalline phase interior; this    locality can offer the distinct advantage of providing a    “biomimetic” milieu for the targeting moiety, a milieu which can    comprise a lipid bilayer as well as hydrophilic domains each of    which can be tuned to optimize the environment;-   2) at the outer surface of the interior—particularly if there is a    distinct phase in between the interior phase and exterior coating,    such as an aqueous layer; such a location could be particularly    advantageous for a particle that would present its targeting moiety    at the new outer surface after release of the exterior coating;-   3) adsorbed to the inner surface of the exterior coating; in this    location, as well as in the other locations listed here, there may    be a synergy between the solid shell and the targeting moiety, in    that certain solid materials (such as aluminum-based compounds, for    example) can sometimes act as adjuvants, to increase the    effectiveness of molecules in the body particularly if they are    meant to interact with the body's immune system;-   4) embedded in the exterior coating, which as discussed above is    most likely to be achievable if the coating is amorphous or at least    semi-crystalline;-   5) at the surface of the exterior coating, either adsorbed or bound    via. e.g., covalent, ionic, hydrogen bonding, and/or hydrophobic    interactions;-   6) attached to, but at a distance from, the surface of the exterior    coating, through attachment via a flexible spacer, e.g., a polymer    that is attached (e.g. by covalently bonding) at one end to a    component of the particle (interior or exterior) and at the other    end to the targeting moiety. Experience with other types of    microparticles in the art has shown that this is generally an    excellent approach for achieving good targeting because it preserves    important conformational and diffusional degrees of freedom that are    sometimes required for good docking of a targeting moiety with a    receptor or target.

It should be noted that in the important case wherein a flexible spacerextends between the targeting moiety and the interior nanostructuredphase, it may be possible to reap the advantages inherent in bothlocations, namely, before dissolution of the coatings the moiety wouldbe in a biomimetic environment provided by the nanostructured interiorphase, and then after dissolution of the coating the moiety would betethered to the (now uncoated) nanostructured phase and thus relativelyfree from hindrance in its interactions with receptors.

Beyond that fact that the interior phases of the instant invention arewell-suited for solubilization of targeting moieties such as proteins,peptides, nucleic acids, polysaccharides, and maintenance of theirconformation, it is also important that many of the lipids, surfactants,and block copolymers which form the basis of many of the embodiments inthe instant invention lend themselves in a very natural way tomodulating the properties of these moieties and their interactions withreceptors in the body. For example, it is known in the art that closeassociation between polyethylene glycol (PEG) chains and proteins orpeptides can have a dramatic effect on stabilizing these peptides, aswell as reducing their degradation by enzymes in the body, in many caseswithout negating their ability to interact with receptors. U.S. Pat. No.6,214,966 (the contents of which are hereby incorporated by reference inentirety) provides examples wherein PEGylation of polypeptides canenhance their performance in the body, including reduced immunogenicityand slower clearance. Furthermore, this effect can be even more dramaticwhen the peptide is associated with a hydrophobic chain (orcholesterol-like group) in conjunction with the PEG chain. U.S. Pat. No.6,309,633 (the contents of which are hereby incorporated by reference inentirety) provides examples of peptides that show greatly increasedstability, resistance to enzymes, and oral absorption when coupled toPEGylated hydrophobic chains or ring systems. Many of the surfactantsand lipids referred to in this specification are PEGylated, or containother oligomeric or polymeric chains that can substantially modify thefate of drugs in the body—or of targeting moieties, as is suggestedhere.

A number of compounds could potentially be used as targeting moieties ina pharmaceutical application of particles of the instant invention. Tobegin with certain lipids, such as Lipid A, have very specificinteractions with components of the immune system, for example, and canbe incorporated into the interior phase or in association with thecoating. Similarly, block copolymers in which one of the blocks couldhave targeting potential, such as glycogen and heparin, may be utilized.Small molecules that could be present either in the interior or exteriorto achieve a degree of targeting include sterols, fatty acids,gramicidin, fragments or simulants of appropriate protein epitopes, andamino acids including aspartic acid, cysteine, tryptophan, leucine andothers. Leucine is an example of a compound that is recognized and boundby a specific protein in the body (the branched-chain amino acidtransporter). Several Examples below describe the production ofparticles coated with leucine.

The ability of the interior phases of the instant invention to providefor solubilization and stabilization of biomolecules, such as thetargeting moieties of focus here, has been described above, where anumber of examples of membrane proteins are given (receptor proteins,such proteins as proteinase A, amyloglucosidase, enkephalinase,dipeptidyl peptidase IV, gamma-glutamyl transferase, galactosidase,neuraminidase, alpha-mannosidase, cholinesterase, arylamidase,surfactin, ferrochelatase, spiralin, penicillin-binding proteins,microsomal glycotransferases, kinases, bacterial outer membraneproteins, and histocompatibility antigens), many of which could serve atargeting role if incorporated in particles of the instant invention.Examples of polymeric components adsorbed to the exterior coating thatcould serve as attachment points for targeting moieties, include, forexample, stabilizing layers on the exterior, i.e., outside the exteriorcoating 20 such as polyelectrolytes or surfactant monolayers (asdiscussed above). The Pluronic F-68 that is used in a number of theExamples is one such polymeric surfactant.

In yet another embodiment of the invention, “externally-directedtargeting” of the coated particles may be achieved. This may beaccomplished by directing particles coated with certain magneticallyresponsive materials discussed above (e.g. ferric oxide) through theapplication of magnetic fields.

Antibodies are broadly useful for targeting to specific sites ormolecules in the body or other environments, and can be incorporated atvarious sites in a particle as discussed above. In particular, intactantibodies with their more hydrophobic Fc fragment are prone topartitioning into matrices of the type used in this invention, andfurthermore it is well known that antibodies can be adsorbed or attached(including covalently) to solid surfaces with retention of binding andbinding specificity. Commercial sources supply antibodies to, forexample, each of the following:

8-hydroxy-guanosine, AAV (adeno virus), ACHE (acetylcholinesterase),ACHER (acetylcholine and NMDA receptor), acid phosphatase, ACTH, Actin(cardiac, smooth muscle, and skeletal), Actinin, Adeno-associated virus,adenosine deaminase, Adipophilin (adipocy differentiation relatedpeptide), Adrenomedulin, 1–6, Advanced glycation end-products (AGE),alanine transaminase, albumin, alcohol dehydrogenase, aldehydedehydrogenase, aldolase, Alfentanil AB, Alkaline Phosphatase, alphaActinin, Alpha-1-anti-chymotrypsin, alpha-1-antitrypsin,alpha-2-macroglobulin, alpha-catenin, beta-catenin and gamma cateinin,Alpha-Fetoprotein, Alpha-fetoprotein receptor, Alpha-Synuclein,Alzheimer Precursor Protein 643–695 (Jonas), Alz-90, Precursor ProteinA4, amino acid oxidase, Amphetamine, amphiphysin, amylase, amylin,Amylin Peptide, Amyloid A and P, Amyloid precursor protein, ANCA(Proteinase PR3), androgen receptor, Angiogenin, Angiopoietin-1 andAngiopoietin-2 (ang-1/Ang-2), Angiotensin Converting Enzyme, AngiotensinII Receptor At1 and At2, Ankyrin, Apolipoprotein D, Apolipoprotein E,arginase I, B Arrestin 1 and B Arrestin 2, ascorbate oxidase,asparaginase, aspartate transaminase, Atpase (p97), atrial NatriureticPeptide, AU1 and AU5, Bacillus Antracis (Anthrax) and Bacill, antracislethal factor, Bad, BAFF, Bag-1, BAX, bcl-2, BCL-X1, B Nerve GrowthFactor, beta Catenin, Benzoylecognine (cocaine), beta-2 microglobulin,beta Amyloid, Galactosidase, beta Glucuronidase, Blood Group antigens(RhoD, A1,A2 A1,A2,A3, B, A, Rh(0)D, RhoC, B M, N), Blood Group Hantigen, bombesin and bombesin/gastrin releasing peptide. BoneMorphogenetic Protein (BMP), Bone marrow stromal cell antigen, BST-3.Borrelia burgdorferi garinii, borrelia burgdorferi sensustricto, BovineSerum, Bradykinin Receptor B2, Brain derived neutrophic factor,Bromodeoxyuridine, CA 19-9, CA 125, CA 242, CA 15-3, CEA, Ca+ ATPase,Calbindin D-28K (Calcium binding protein), Calgranulin A, Cadherin,CD144, Calcineurin, Calcitonin, Calcitonin gene related peptide, CalciumChannel, Caldesmon, Calmodulin, Calnexin, Calpactin light chain,Calpain, Calpastatin, Calreticulin, Calretinin, Calsequestrin, CamKinase II, Canine Distemper virus, carbonic anhydrase I and II,Carboxypeptidase A, B and E, Carboxypeptidase Y, Cardi, Troponion C andT, cardiotrophin-1, Caspase 3 (CPP32), Catalase, Catetins, Caveolin 1, 2a and 3, CCR, CD44 (HCAM), CD56 (NCAM), CDK2, CDK4 (Cyclin DependentKinase C), Carcinoembryonic Antigen, Cellular antigens, CFTR (cysticfibrosis transmembrane conductance protein), chemokine receptors,chlamydia, CHO cell (Chinese Hamster Ovary Cell) Proteins, choleratoxin, choline oxidase, Chondroitin, Chloramphenic,Acetyltransferase(CAT). Chromagranin A, B and C (Secreogranin III),cholesterol oxidase, Chymotrypsin, Cingulin, Citrate Synthethetase,C-kit/stem cell factor receptor, CK-MB, Clathrin Antigen, ClostridiumBotulinum D Toxoid, Clusterin, C-MYC, CNS Glycoprotein 130 kD, CollagenType IV and Type VII, Complement 5b neoepitope, Complement C3a, C3b, C5and C9, complexin 2, Corticoliberin (CRF), C-peptide, CRF (CorticotropinReleasing Factor), Corticotropin releasing factor receptor, COX-1 andCox-2, CPP32 (also known as Caspase 3, apopain or Yama), Creatinetransporter, C-Reactive Protein (CRP), Cryptosporidium, CXCR-5, CyclinA, Cyclin D1, D2 and D3, Cyclosporine A, Cylicin I, Cytochrome B5,Cytochrome C, Cytochrome oxidase, Cytochrome P450, Cytokeratin Types Iand II, Cytomegalovirus, DAP Kinase, Dendritic cells, Desmin,Desmocollin 1, 2 and 3, Desmoglein 1, 2 and 3, Desmoplakin 1 and 2,Dextranase, DHT (Dihydrotestosterone), Dihydrofolate Reductase (DHFR),Dioxin, Diptheria toxin, Distemper, DJ-1, DNA single-stranded, DNAdouble stranded, DNA Topoisomerase II and Phospho-topoisomerase IIa+IIalpha/beta, Dopamine, Dopamine Beta-Hydroxylase, Dopamine Receptor,Dopamine Transporter, Drebrin, Dysferlin, Dystrobrevin, E. Coliexpression plasmid, Elastase, Elastin, Endocrine Granu, Constituent(EGC), Endorphin, Endothelial cell, Endothelin, Endothelin Receptor,Enkephalin, enterotoxin Staphylococcus aureus, Eosinophil Peroxidase,Eosinophil derived neurotox, (EDN), Eotaxin, Eotaxin-2, Epidermal GrowthFactor, Epidermal Growth Factor 2, epidermal growth factor receptor,testosterone, Epithelial Proliferating antigen, Epithelium SpecificAntigen, c-MYC, HA.1, VSV-G Tag, Glu-Glu, EEEYMPME, Thioredoxine (trx),Epstein Barr virus and Epstein Barr Virus capsid antigen gp 120, ERK(ERK1, ERK2, ERK3, pan ERK also called MAP kinase), Erythrocytes,Erythropoietin (EPO), Esterase, Estradiol, Estriol, Estrogen Receptor,Estrone, Ets-1 transcription, F1 antigen Yersina pestis, Factor 5,Factor VII, Factor VIII, Factor 9, Factor 10, Factor 11, Factor 12,Factor XIII, FAK (Focal Adhesion Kinase), FAS (CD95), FAS-L (CD178),Fascin, Fatty Acid Binding Protein, Ferritin, Fetal Hemoglobin,Fibrillin-1, Fibrinogen, Fibroblasts, Fibroblast Growth Factor, FGF-9,Fibronectin, Filamin, FKBP51, FKBP65, FK506, FLK1, flt-1 FLt-4 andFLT-3/FLK-2, FLT 3 Ligand, Fluorescein (FITC), FODRIN, Folate, FolateBinding Protein, fractaikine, frequenin, Frizzled, Fructose-6-p-kina,FSH, Fusin (CXCR4), GABA A and GABA B Receptor, Galectin, galanin,gastrin, GAP-43, G-CSF, G-CSF receptor, gelsolin, GIP (gastricinhibitory peptide), G0-protein (bovine), GDNF, GDNF-Receptor, Giardiaintestinalis, Gliai fibrillary acidic Protein, Glial filament protein,Glucagon/Glycentin, Glucose oxidase, Glucese 6 Phosphate Dehydrogenase,Gluco, Tranporter GLUT 1–4, GLUT 1–5, Glutamate Dehydrogenase, GlutamicAcid decarboxyla (GAD), Glutathione, (Glyceraldehyde-3-phosphatedehydrogenase GAPDH, Glycerol-3-phosphate dehydrogenase, Glycerolkinase, glycine transporter (GLYT1, GLYT2), Glycogen PhosphoralaseIsoenzyme BB (GPBB), Glycophorin A (CD235a), GM-CSF, C receptor alpha,Golgi Complex, Gonadotropin-Releasing Hormone Receptor (GnRHR), GP130,Granzyme, GRB2, GRB1, Green Fluorescent Protein (GFP), Growth Hormone,Growth Hormone Receptor, Growth Hormone Releasing factor, GRP78,Hantavirus, HCG, HDL (high density lipoprotein), Heat Shock ProteinHSP-27, HeK 293 Host Cell Proteins, Helodermin, helospectin,Hemeoxygenase, Hemoglobin, Heparin, Hepatitis A, Hepatitis B CoreAntigen, Hepatitis B virus surface antigen, Hepatitis C virus,Hepatistis E virus, Hepatitis G Virus, Hepatocyte Growth Factor,Heregulin (Neu differentiation factor/Neuregulin), Herpes Simplex Virus,Hexokinase, Histamine, His Tag, 6-His vector tags, HIV-1 p24, p55/17,gp41, gp120, tat, nef, rev, HIV reverse transcriptase, HLA Class I, HLAClass II, HLA-DM, HLA DQw1, HLA DRw 52, Peroxidase, HPV 16 Late IProtein, human free kappa light chains, human lambda light chains, HumanIgA, human I heavy chain, human IgA1, human IgD, human IgE, human IgGheavy chain, human IgG1, human IgG3, human IgG4, human IgM, human IgMheavy chain, human J chain, human kappa lig, chains, human lambda lightchains, Human Serum Amyloid P. Human Serum Amyloid P, Interleukin 1 betaconverting enzyme, ICH-1 (caspase 2), Indian Hedgehog Protein (IHH),Influenza virus, Inhibin, Insulin, insulin like growth factor II,insulin growth factor binding protein 1, 2, 3, 4 or 5, insulin likegrowth factor, insulin like growth factor I receptor, insulin receptor,insulin/proinsulin, Interferon alpha, interferon alpha receptor.Interferon beta, Interferon gamma, interferon gamma receptor alpha andbeta, Interleukin 1 alpha, Interleukin Receptor alpha type II,Interleukin 1-beta, Interleukin 10, interleukin 10 receptor, Interleukin11, Interleukin 12, interleukin 12 receptor, Interleukin 13, Interleukin15, Interleukin 16, Interleukin 17, Interleukin 18, Interleukin 2,Interleukin 2 receptor alpha, Interleukin receptor alpha chain (CD25),Interleukin 2 receptor beta, Interleukin 2 receptor beta chain(CD122),Interleukin 2 receptor gamma, Interleukin 3, Interleukin 3/interleukin5/GM-CSF Receptor common chain, Interleukin 4, Interleukin 5,Interleukin 6, Interleukin 6 receptor alpha chain, Interleukin 7,Interleukin 7 receptor alpha, Interleukin 8, Interleukin 8 receptor,Interleukin 9, invertase, Involucrin, IP-10, Keratins, KGF, Ki67,KOR-SA3544, Kt3 epitope tag, lactate dehydrogenase, Lactoferrin,lactoperoxidase, Lamins, Laminin, La (SS-B), LCMV (LymphocyticChoriomeningitis Virus), Legionella pneumophilia serotype, Legionellapneumophila LPS, Leptin and Leptin Receptor, Lewis A Antigen, LH(leutenizing Hormone), LHRH (leutenizing Hormone Releasing), L,(leukemia Inhibitory Factor), 5-Lipoxygenase, LPS Francesellatularensis, luciferase, Cancer Marker (MOC-1, MOC-21, MOC-32, Moc-52),Lymphocytes, lymphotactin, Lysozyme, M13, F1 Filamentous Phages,Macrophages/monocytes, Macrophage Scaveng, Receptor, Matrixmetalloproteases, M-CSF, Major Basic Protein, malate dehyrogenase,Maltose Binding Protein, Mannose Receptor (macrophage),Mannose-6-phosphate receptor, MAP kinase antibodies (ERK, ERK, ERK2,ERK3), MASH1 (Mammalian achaete schute homolog 1 and 2), MCL-1, Mcm3, M,(MCAF), MCP-2, MCP-3, Melanocortin Receptors (1 through 5), Met (c-met),Mineralcortocoid Receptor (MR/MCR), Melanoma Associated Antigen, MGMT(methylguanine-DNA-methyltransferase), MHC Antibodies (incl. HLA DATAPACK), Milk F, Globule Membrane, Milk Mucin Core Antigen, MIP-1 alpha,MIP-1 beta, Mitochondrial markers, Mitosin, MMP-1, MM, MMP3, MMP7, MMP8,MMP-9 and MMP13 (matrix metalloproteases), MMP-14(MT1-MM, MMP15(MT2-MMP), MMP16(MT3-MMP) and MMP19, Morphine, motili, Mucin relatedantibodies (Muc-1, muc-2, muc-3, muc-5ac), Mucin-6 glycoprotein,Mucin-like Glycoprotein, Mycobacterium tuberculosis, Myelin, MyelinBasic Protein, Myeloperoxidase, MyoD, Myoglobin, Myosin, Na+ Ca+Exchanger Protein, Na+/K+/ATPase, Na+/K+/ATPa, NCAM (CD56), pan N-Cam,(neural cell adhesion marker), Nerve Growth Factor, Neu-Oncogene (c-erbB2), Neurofibrillary Tangle, Neurofilament 70+200 kD, Neurofilament145Kd, neurofilament 160kd. Neurofilament 68Kd, Neurofilament 200kd,Neurofilament 200kd, neurokin, A/substance K, neuromedin U-8 (NMU-8),Neuromodulin, neuronal pentraxin, Neuro-Specific Enolase, Neuropeptide Y(NPY), Neurophysin I (oxytocin precursor), Neurophysin, (vasopressinprecursor), Neuropsin, Neurotensin, NFKB, Nicotinic AcetylcholineReceptor, (Beta2 and Alpha 4), NMDA receptors, N-MYC, NorepinephrineTransporter (NET), N, (Nitric Oxide Syntase) eNos, iNos, NT-3, NT,(neurotroph. 4), Nucleolar Helicase, Nucleolar Protein N038, NuclearProtein xNopp180, Nucleoplasm, Protein AND-1, Nucleolus OrganizingRegion (NOR), Nucleolin, occludin, Oncostatin M, ORC, OrnithineDecarboxylase, Ovalbumin, Ovarian Carcinoma, Oxytocin, P15, P16, P2,P27, P53 Oncoprotein, p62 Protein, p97 Atpase, membrane associated andcytosolic 42 kDa inositol (1,3,4,5) tetrakisphosphate receptor, PP44Podocyte Protein (Synaptopodin), PAH (Polyaromatic Hydrocarbons), PACAP(pituitary adenylate cyclase activating peptide), Pancreas Polpeptide(PP), Pancreastatin, Pancreatic Islet Cell, papain, Papillomavirus(HPV), Parainfluenza type 2 viruses, Parathion, Parkin, PARP (Poly-A,Riobose Polymerase) PARP-1 and PARP-2, Patched-1, Patched-2, Paxillin,polychlorinated biphenyls, Pemphigus vulgaris (desmoglein 3),Penicillin, penicillinase, pep-carboxylase, pepsin, Peptide YY, Perforinand polyclonals, Perilipin, Peripherin, Perlecan, Petrole, Hydrocarbons(total), PPAR (peroxisome proliferation activated receptors),P-Glycoprotein (multi-drug resistance), PGP9.5, Phenanthrene,Phencyclidine (PCP), Phenylethanolamine, methyltransferase (PNMT),Phospholamban, Phospholipase A2, Phosphoserine, Phosphothreonine,Phosphotyrosine, Phosphothreonine-proline, phosphothreonine-lysi,phophotyrosi, Phosphotyrosine Kinase, Pichia pastoris, PlacentalAlkaline Phosphatase, Plakoglobin, Plakophilin 1, Plakophilin 2,Plakophilin 3, Plasminogen, Platelet Derived Growth Factor AA and BB andAB, Plectin, PM, ATPase (plasma membrane Ca pump), Pneumocystis carinii,Pneumolysin, Polychlorobiphenyl (PCB), PP17/TIP47, PPAR (peroxisomeproliferation activated receptors), Prednisone, Prednisolone, Pregnancyassociated Plasma Protein A (PAPP-A), Pregnenolone, Prepro NPY 68–97,Presenilin-1, Presenilin-2, Prion protein, Progesterone, Progestero,Receptor, Prohibitin, Proinsulin, Prolactin, Proliferation Ce, NuclearAntigen, Proline Transporter, Prostatic Acid Phosphatase (PAP),Prostatic Specif, Antigen (PSA), Proteasome 26S, Protein 4.1 M ascites,Protein G, Protein Kinase C. Pseudomonas mallei, PTH, PulmonarySurfactant Associated Proteins, Puromycin, Pyruva, kinase, Rabies Virus,RAC-1 Rac-2, RAGE (receptor for AGE), RANTES, RDX, RecA, Receptor foradvanced glycation end products (RAGE), Red Blood cells, Regulatorysubunit, RELM alpha and Beta (resistin like molecules), Renin, Rennin,Replication Protein A (RPA p32 and p70), Resistin, Respiratory syncytialvirus (RSV), Retinoblastoma (Rb), phospho-specific RB (ser780),Ribonuclease A, RNA Polymera, Arna3, RNP (70KdaU1), A Protein, BProtein, RO (RO52, Ro60), Rotavirus group specific antigen, Rubellavirus structural glycoprotein E1, Ryanodine Receptor, S-100 Protein,saccharomyces cerevisiae, Salmonella O-antigens, Salmonel, typhimurium,Sarcosine Oxidase, SDF-1 Alpha and SDF-1 Beta, secretin, SelenoproteinP, Serotonin, Serotonin Receptor, Serotonin Transporter, Sex HormoneBinding Globulin (SHBG), SFRP5 (secreted frizzled-related protein 5),SF21 and SF9, SIV gp120, SIV p28, Smooth muscle actin, Somatostatin,Staphylococcus aureus, Staphylococcus aureus enterotoxin, STAT1, Stat2,Stat, Stat4, Stat5 Stat6, Stem Cell Factor (SCF) and SCFR/C-kit,Streptavidin, Streptococcus B, Stromal Cell Derived Factor-1 (SDF-1alpha and beta), Substance P, Sufentanil AB, Superoxide Dismutase,Surfactant Associated Proteins (A,B,C,D), Symplekin, Synapsin I,Synapsin IIa, Synaptophysin, Synaptopodin (Podocyte Protein), Syndecan1, Synphilin-1, Synuclein (alpha), SV40 Large T antigen and small Tantigen, Talin, TARC, TAU, Taurine transporter, Tenascin, Testosterone,TGF-alpha, TGF-beta, TGF beta receptor (Endoglin), THC, ThomsenFriedenreich Antigen (TF), THY-1 25kd Brain (CDw90), Thymocytes,Thrombin and Thrombin Receptor, Thyroglobulin (24TG/5E6 and 24Tg/5F9),Thyroid Binding Globulin, Thyroid Hormone Receptors, Thyroid Peroxidase,Thyroid Stimulating Hormone (TSH), Tyrosine Hydroxylase, ThyrotropinReleasing Hormone (TRH), Thyroxine (T4), TIe-1 and TIe-2, TIMP-1,TIMP-2, TIMP-3 (Tissue Inhibitors, metalloproteinase), Titin, TNFreceptor associated factors 1 and 2, TNF Receptor, TNF receptor II,TNF-Alpha, TNF-Alpha, TNF-beta, Toxoplasma gondii p30 antigen, TPO(thrombopoietin), TRAF, Traf2, Traf3, TRAF4, TRAF5, TRAF6, Transferrin,Transferrin Receptor, Transforming Growth Factor A, Transformi, GrowthFactor Beta, Transportin, Trepone, pallidium, Triiodothyronine (T3),Trinitrotoluene (TNT), TRK A, TRK B, TRK C, Tropon, (cardiac), TroponinI, Troponin T, trypsin, trypsin inhibitor, trypsinogen, TSH, TUB Gene,Tubulin alpha and beta, Tubulin beta specific, Tumor Marker relatedAntibodies, Tumor Necrosis Factor Alpha, Tyrosinase, Tweak, (caspase-4),Ubiquitin, Ubiquitin-L1, Uncoupling Proteins (UCP1, UCP2, UCP3, UCP 4and UCP5), Urease, Uricase, Urocortin, Uroplakin, Vasopressin,Vasopressin Receptor, VEGF, Vesicular acetychoine transport, (VACht),Vesicular monoamine transporter (VMAT2), Villin, Vimentin, Vinculin, VIP(Vasoactive Intestinal Peptide), Vitamin B12, Vitamin B12, Vitamin Dmetabolites, Vitamin D3 Receptor, Von Willebrand Factor VSV-G EpitopeTag, Wilm's tumor Protein X, Oxida, Yeast, hexokinase, SOD, cytochromeoxidase, carboxypeptidase, and Yersinia eterocolotica.

Alternatively many of the substances noted above (e.g. folate, PGP,cytochrome P 450, and EGF) may in and of themselves be useful astargeting substances and may be incorporated into the particles of thepresent invention. In addition, other chemical compounds such as PEG mayalso be used for targeting and may be incorporated.

It is important to point out that in addition to targeting compounds perse, active compounds, functional excipients such as absorptionenhancers, and other bioactive materials as gleaned from the lists ofmaterials given herein can be incorporated in any of these localizationsites.

In addition to the targeting of particles to specific sites for releaseof drug, as mentioned above particles incorporating certain radiopaqueor optically dense materials could themselves be used for imaging, andwhen coupled to targeting compounds as described herein could targetspecific sites in the body and allow their visualization. As an example,somatostatin receptors are known to be localized at certain tumor sites,so that the attachment of a target to coated particles as per theinstant invention that would bind selectively to somatostatin receptorscould target a tumor and allow visualization via, e.g., x-ray, MRimaging, or radioimaging. To extend this idea, a similarly targetedparticle could then carry a radioactive material that would emitradiation intended to induce necrosis of the tumor.

Polymerized Liquid Crystals as Interior Phases.

U.S. Pat. No. 5,244,799 (the contents of which are hereby incorporatedby reference in entirety) reports the polymerization of nanostructuredcubic and hexagonal phase liquid crystals, with retention of theirnanostructure. The retention of structure was demonstrated bysmall-angle x-ray scattering (SAXS) and transmission electron microscopy(TEM).

The possibility of polymerizing the cubic phase in the interior of aparticle of the instant invention opens up a number of possibilities,partcularly as relate to increasing the stability of the interior phaseand modulating its interaction with the body, and cell membranes inparticular. For an example of the latter, whereas an unpolymerized cubicphase might be expected to molecularly disperse when coming into contactwith a biomembrane, polymerization of the same interior matrix mightcreate a particle interior that would retain its integrity throughoutits interaction with the same biomembrane, and this could have dramaticconsequences as to the fate of the particle and to a drug inside theparticle. Furthermore, the retention of a bilayer-bound drug(hydrophobic small molecule, membrane protein, etc.) might be increasedtremendously by polymerization, yielding a slow-release particle. Andthe presence of a more permanent, precisely-defined pore structure, withprecisely tunable poresize, might make possible improved controlledrelease of a drug, and/or sequestration of the drug from degradative orother enzymes by size-exclusion from the pores of the polymerizedmatrix.

The following examples illustrate the present invention but are not tobe construed as limiting the invention.

EXAMPLES

In the following examples, Examples 14, 15, 16, and 34 demonstratesystems with coatings made of physically robust mineral materials, suchas cupric ferrocyanide and calcium phosphate, that can provide forstability of the intact particles under stronger shear conditions, suchas during pumping of a dispersion of the coated particles, for example,for recycling or transport. These minerals are also of low aqueoussolubility, making them of potential interest in applications requiringrelease of the particle coating by strong shear, while at the same timeprotecting against release due to simple dilution with water. An exampleof such an application would be where a rodent deterrent such ascapsaicin, or rodent toxin, would be encapsulated in the coatedparticles of the present invention, the particles impregnated intoelectrical wires, corrugated boxes, and other products requiringprotection against pawing by rodents, and the pawing action of a rodentwould induce release of the active deterrent or toxin. The low watersolubility would prevent the deterrent from premature release due todamp conditions.

A robust organic material that provides a coating that is also of lowaqueous solubility is ethylhydrocupreine, as in examples 17 and 33, andthis compound has the additional characteristic that it has an extremelybitter taste that could provide an additional deterrent effect in arodent-deterrent application.

Examples 1, 2, 3, 6, 7, 8, 9, 10, 17, 18, 19, 20, 23 and 33 provideexamples of coatings that are of low water solubility at neutral pH, butthat increase substantially in solubility as the pH becomes eitheracidic or basic, depending on the compound. This can make the coatedparticles of importance in, for example, drug delivery, where a coatingthat releases preferentially in a particular pH range is desired, suchas for intestinal release. Or such a coating could release, allowing therelease of an antibacterial compound, at sites of bacterial activity,where pH is typically acidic. Or the release of the coating at aparticular pH could allow the release of a pH stabilizing compound or abuffer system, for example in microparticles designed to control the pHof water in swimming pools.

Example 4 gives an example of particles with a coating, silver iodide,that could provide very useful properties as a cloud-seeding agent,since the silver iodide coating is well-known for cloud-seedingeffectiveness, and the surface area and surface morphology afforded bythe particle shape and size could amplify the effect of the silveriodide. This could be of commercial importance due to the expense ofsilver compounds, in which case the inexpensive liquid crystal interiorcould serve the role as a filler that would provide the same or greaterseeding potential at a fraction of the cost of simple silver iodide. Asimilar increase in effectiveness due to amplification of surface areamight prove of interest in the use of the particles as local anestheticsfor mucous membranes, and the proper balance of lipids and activeanesthetic hydrophobes (such as lidocaine) in the particle interiorcould be used to enhance the effect.

Example 5 demonstrates that compounds such as sulfides and oxides can beused as coatings in the coated particles of the present invention, evenwhen they require gaseous reactants for formation. Such compounds arewell-known for being not only high-stiffness materials, but alsochemically extremely resistant, which could make such coated particlesof interest in applications where the particles encounter harsh chemicaland physical conditions, such as would be expected in use of theparticles as polymer additives, or in processing involving high shear,such as impregnation of dye-containing particles in nonwoven materials,etc.

Examples 12 and 13 demonstrate the use of high water-solubilitycompounds as coatings, that can be of importance in applicationsrequiring quick and convenient release of the coating by simple dilutionwith water. For instance, a spray system that would merge two streams,one containing the dispersion and the other water, could provide anaerosol in which the particle coatings, useful for preventingagglomeration prior to spraying, would be dissolved after spraying whenthe particles were already aerosolized—in flight, so to speak. Sincethis dissolution could expose, for example, a nanostructured cubic phaseinterior that was very tacky, the particles could be used to adhere to,e.g., crops, or the bronchial lining, etc. The capsaicin loaded into theinterior in Examples 12 and 13 would make this product of potentialimportance in providing rodent resistance after deposition of the tackyaerosolized particles onto crops, for example, since rodents aregenerally strongly repelled by the taste of capsaicin even at very lowconcentrations.

Examples 39 and 40 demonstrate the use of amorphous materials asexterior coatings for the particles of the present invention. Example 39utilizes the amorphous polymer (PLGA), and Example 40 utilizes a smallmolecule (the sugar trehalose).

Examples 41 and 42 demonstrate variations in the processes used tocreate particles, and in particular they involve processes in whichmilling or particle size reduction are applied after formation of thecoating material, in some cases in addition to sonication ormicrofluidization that is applied prior to coating materialprecipitation.

Examples 1 (part E), 27, 28 and 43 demonstrate the incorporation ofactive targets, including receptors, lectins, and antibodies, inparticles according to the instant invention, and the retention of theirbinding capabilities.

All percentages in the following examples are weight percentages unlessotherwise noted. The amounts of the components used in the followingexamples can be varied as desired, provided that the relative amountsremain as in the example: thus, these amounts can be scaledproportionately to the desired amount, recognizing of course thatscaling up to large amounts will require larger equipment to process.

In the following examples, unless otherwise noted, the exterior coatingof each coated particle comprises a nonlamellar material, and eachinterior core comprises a matrix consisting—essentially of at least onenanostructured liquid phase, at least one nanostructured liquidcrystalline phase or a combination of at least one nanostructured liquidphase and at least one nanostructured liquid crystalline phase.

Example 1

This Example shows that a wide range of active compounds, includingcompounds of importance in pharmaceutics and biotechnology, can beincorporated into nonlamellar material-coated particles of the presentinvention.

An amount of 0.266 grains, of sodium hydroxide was dissolved in 20 ml ofglycerol using heating and stirring to aid in dissolution. An equimolaramount, namely 1.01 grams, of methyl paraben was then dissolved, againwith heating. From this solution, 0.616 grams were taken out and mixedwith 0.436 grams of lecithin and 0.173 grams of oleyl alcohol in a testtube. The active ingredient (or agent) identified below was incorporatedat this point and the solution thoroughly mixed to form a nanostructuredliquid crystalline phase material with an active ingredient disposedwithin it. An “upper solution”, which was obtained by dissolving 0.062grams of Pluronic F-68 (a polypropyleneoxide-polyethyleneoxide blockcopolymer surfactant commercially available from BASF), and 0.0132 gramsof acetic acid together and adding to the test tube as a layer ofsolution above the previous solution that included the active agent.Immediately the test tube containing the liquid crystalline mixture andthe upper solution was shaken vigorously and sonicated for three hoursin a small, table-top ultrasonicator (Model FS6, manufactured by FisherScientific). The resulting dispersion showed a high loading of particlescoated with methyl paraben with size on the order of one micron uponexamination with an optical microscope.

Example 1A 2.0 wt % of salicylic acid (based on the weight of theinternal core of liquid crystalline phase material) was incorporated asan active agent.

Example 1B 2.0 wt % Vinblastine sulfate (based on the weight of theinternal core of liquid crystalline phase material) was incorporated asan active agent.

Example 1 C 2.4 wt % Thymidine (based on the weight of the internal coreof liquid crystalline phase material) was incorporated as an activeagent.

Example 1 D 1.6 wt % Thyrotropic hormone (based on the weight of theinternal core of liquid crystalline phase material) was incorporated asan active agent.

Example 1 E 2.9 wt % Anti-3′, 5′cyclic AMP antibody (based on the weightof the internal core of liquid crystalline phase material) wasincorporated as an active agent.

Example 1 F 2.0 wt % L-Thyroxine (based on the weight of the internalcore of liquid crystalline phase material) was incorporated as anactive.

Particles such as these with a coating which increases substantially insolubility as the pH increases, could be useful in drug delivery, wherethe increase in pH moving along the gastrointestinal tract from thestomach to the intestines could result in effective delivery to thelower gastrointestinal tract, giving rise to a more uniform deliveryrate over time.

Example 2

This example demonstrates the long-term stability of a dispersion ofparticles of the present invention.

The amino acid D,L-leucine, in the amount of 0.132 grams, was dissolvedin 2.514 grams of 1 M hydrochloric acid, resulting in the formation ofleucine hydrochloride in solution. The solution was dried on a hot plateunder flow of air, but was not allowed to dry to complete dryness:drying was stopped when the weight reached 0.1666 gram, whichcorresponds to one molar equivalent addition of HCl to the leucine. Anamount of 0.130 grams of this compound were added to 0.879 grams of ananostructured reverse bicontinuous cubic phase material prepared bymixing sunflower oil monoglycerides and water, centrifuging, andremoving the excess water. An upper solution was prepared by mixing 1.0grams of 1 M sodium hydroxide with 3 grams of water. All water used wastriply-distilled. The upper solution was overlaid on the cubic phase,the test tube sealed and sonicated, resulting in the formation of amilky-white dispersion of microparticles coated with leucine.

A similar dispersion was prepared with the use of Pluronic F-68 asstabilizer. An amount of 0.152 grams of leucine hydrochloride was addedto 0.852 grams of nanostructured reverse bicontinuous cubic phasematerial as above, and an upper phase consisting of 0.08 grams of F-68,1.0 gram of 1 M sodium hydroxide, and 3.0 grams of water was overlaid onthe nanostructured reverse bicontinuous cubic phase material andsonicated. Again, a milky-white dispersion of leucine-coatedmicroparticles was formed, where this time the F-68 amphiphilic blockcopolymer surfactant coated the outer (leucine-based) surface of theparticles.

As a control experiment to show the necessity of the leucine for theformation of crystalcoated panicles, 1.107 grams of Dimodan LS(hereinafter “sunflower monog lycerides) were mixed with 1,000 gram ofwater to form a nanostructured reverse bicontinuous cubic phasematerial. An upper solution was prepared by adding 0.08 grams ofPluronic F-68 to 4.00 grams of water. As per the same procedure used tomake the dispersions above using leucine, the upper solution wasoverlaid on the nanostructured reverse bicontinuous cubic phase materialand the test tube sealed and sonicated. In this case, essentially nomicroparticles were formed: the nanostructured reverse bicontinuouscubic phase material remained as large, macroscopic chunks even afterseveral hours of sonication under the same conditions as the leucineexperiment.

This dispersion of the coated particles of the present invention wasexamined regularly for a period of twelve months and did not show signsof irreversible flocculation. With even slight agitation, it showed nosigns of irreversible flocculation over time scales of weeks. In theabsence of agitation, it did show signs of flocculation, but upon mildshaking for 5 seconds or more, any flocculation reversed. A droplet ofthe dispersion was examined in an Edge Scientific R400 3-D) microscopeat 1,000 magnification (100× objective, oil immersion, transmittedlight) and shown to have a very high loading of submicron particles.

Particles such as these, with relatively weak organic coatings, can beused, for example, in acne creams, where an active material such astriclosan could be incorporated and the shear associated with applyingthe material to the skin would release the coating.

Example 3

In this example paclitaxel was incorporated at the level of 0.5% of theinternal core. The particle coating was leucine, which in other examplesherein has been shown to provide longterm stability.

A paclitaxel-containing nanostructured reverse bicontintuous cubic phasematerial was produced by mixing 4 mg of paclitaxel, dissolved in 2 mlt-butanol, in a nanostructured reverse bicontinuous cubic phase materialcontaining 0.280 gm lecithin, 0.091 gm of oleyl alcohol, and 0.390 amglycerol: after evaporation of the butanol under argon, a nanostructuredreverse bicontinuous cubic phase material formed that was viscous andoptically isotropic. The sample was centrifuged for one hour, duringwhich time no precipitate appeared. Optical isotropy was verified in apolarizing optical microscope. A leucine hydrochloride solution inglycerol was produced by mixing 0.241 grams of leucine, 2.573 grams of 1M HCl, and 0.970 grams of glycerol, after which the water and excess HClwere evaporated under air flow on a 50° C. hot plate, drying for threehours. Next, 0.882 grams of this leucine-HCl in glycerol solution wereadded to the nanostructured reverse bicontinuous cubic phase material.The upper solution was then prepared by adding 0.102 grams of PluronicF-68 to 4.42 grams of an aqueous buffer at pH 5.0. After overlaying theupper solution onto the nanostructured reverse bicontinuous cubic phasematerial, the nanostructured reverse bicontinuous cubic phase materialwas dispersed into microparticles by sonicating for 2 hours.

Particles such as these could be used for the controlled release of theantineoplastic agent paclitaxel.

Example 4

In this example, the coating was silver iodide, which has the potentialto make the particles useful in photographic processes. Silver iodide issomewhat unusual in that, even though it is a simple salt (withmonovalent ions only), it has a very low solubility in water. Ananostructured reverse bicontinuous cubic phase material was prepared bymixing 0.509 grams of Dimodan LS (commercially available as fromGrinstedt AB and referred to herein as “sunflower monoglycerides”),0.563 grams of triply-distilled water, and 0.060 grams of sodium iodide.An upper solution was prepared by adding 0.220 grams of silver nitrate,0.094 grams of Pluronic F-68, and 0.008 grams of cetylpyridiniumchloride to 3.01 grams of water. A dispersion of microparticles was thenproduced by overlaying the upper solution onto the nanostructuredreverse bicontinuous cubic phase material and sonicating for one hour.The particle coating was silver iodide, which has a low solubility inwater.

Example 5

In this example cadmium sulfide was used as the coating. It is itnonlamellar crystalline compound that exhibits large changes in physicalproperties when doped with small amounts of other ions. This examplealso demonstrates that a gas, such as hydrogen sulfide gas, can be usedin the present invention to induce crystallization and particleformation.

A nanostructured reverse bicontinuous cubic phase material was preparedby thoroughly mixing 0.641 grams of Dimodan LS with 0.412 grams ofwater, and to this was added 0.058 grams of cadmium sulfate hydrate.After this, 0.039 grams of calcium sulfide was overlaid on the mixture,and the test tube was purged with argon gas and capped. An uppersolution was prepared by adding 0.088 grams of Pluronic F-68 and 1.53grams of glycerol to 1.51 grams of 1M HCl and then sparging the solutionwith argon. The upper solution was taken up in a syringe, and added tothe first test tube. Upon addition, the smell of hydrogen sulfide gascould be detected in the test tube, as well as the formation of ayellowish precipitate: this indicated the action of hydrogen sulfide gasin producing cadmium sulfide (CdS) from the cadmium sulfate. The systemwas sonicated, resulting in a dispersion of microparticles which had acadmium sulfide coating.

Example 6

This example demonstrates that the interior is substantially protectedfrom contact with conditions outside the particle by the crystallinecoating, which here is leucine. Any contact with zinc dust chancesmethylene blue to colorless in less than one second; here, addition ofzinc did not cause a loss of color for some 24 hours. Although there wasan eventual loss of color, that loss is believed to be due simply to theeffect of the zinc on the leucine coating.

A solution of leucine hydrochloride in water was made by mixing 0.122grams of leucine with 1.179 grams of 1M HCl and evaporating untilapproximately 1 gram of solution remained. To this was added 0.922 gramsof sunflower monoglycerides, and 10 drops of a strongly colored aqueoussolution of methylene blue. An upper solution was produced by adding0.497 grams of 1M NaOH and 0.037 grams of Pluronic F-68 to 3.00 grams ofpH 5 buffer. The upper solution was overlaid, the system sonicated, anda dispersion of microparticles formed. An aliquot of the dispersion wasfiltered to remove any undispersed liquid crystal, and 0.1 grams of 100mesh zinc dust added. (When zinc dust is shaken with a solution ofmethylene blue, the reducing effect of the zinc removes the blue color,normally in a matter of a second, or almost instantaneously.) However,in the case of the microencapsulated myethylene blue produced by thisprocess, it took on the order of 24 hours for the color to disappear,finally resulting in a white dispersion. Thus, despite interactionsbetween the zinc and the leucine that can disrupt the coatings of theseparticles, the coatings provided substantial protection of the methyleneblue against the effect of the zinc, increasing the time required forzinc reduction of the dye some 4–5 orders of magnitude.

If particles such as these are employed in a product in which two activeingredients must be sequestered from contact with each other (such asthe oxidation-sensitive antibacterial compound triclosan and thestrongly oxidizing cleansing agent benzoyl peroxide), this experimentdemonstrates the feasibility of using leucine-coated particles inpreventing contact between an encapsulated compound and the environmentoutside the particle.

Example 7

In this example a leucine coating protects the methylene blue dye in theparticle interior from contact with ferrous chloride, as easily seen bythe absence of the expected color change when ferrous chloride is addedto the dispersion. This indicated that the coating was substantiallyimpermeable even to ions.

A solution of leucine hydrochloride in glycerol was made by mixing 0.242grams of leucine, 2.60 grams of 1 M HCl, and 1.04 grams of glycerol, andthen drying on a 50° C. hot plate under flow of air for 1.5 hours. Ananostructured reverse bicontinuous cubic phase material was prepared bymixing this leucine-HCl solution, 0.291 agams of lecithin (Epikuron 200,from Lucas-Meyer), 0.116 grams of oleyl alcohol, and 0.873 grams ofglycerol; this was colored by the addition of a pinch of methylene blue.An upper solution was prepared by adding 0.042 grams of Pluronic F-68surfactant to 4.36 grams of pH 5 buffer, overlaid on the nanostructuredreverse bicontinuous cubic phase material, and the system sonicated toproduce a dispersion of microparticles. To an aliquot of this dispersionwas added 0.19 grams of ferrous chloride, a reducing agent. The absenceof a color change indicated that the methylene blue was protectedagainst contact with the ferrous compound by encapsulation in theleucine-coated particles, since the addition of ferrous chloride tomethylene blue solutions normally changes the color to blue-green(turquoise).

Similarly to Example 6, this experiment shows that encapsulatedcompounds such as methylene blue which are sensitive to, in this case,reducing agents, can be protected against reducing conditions outsidethe particle until release of the coating. This could be useful in, forexample, electrochemical applications where the effect of application ofelectrical current would be gated by the chemical release of thecoating.

Example 8

This example, when considered along with Example 1A and Example 10,demonstrates that particles of the present invention coated with methylparaben can be produced in two entirely different ways: either by athermal process, such as a heating-cooling method, or by a chemicalreaction, such as an acid-base method.

To a nanostructured reverse bicontinuous cubic phase material, producedby mixing 0.426 grams of sunflower monoglycerides (Dimodan LS) with0.206 grams of acidic water at pH 3, were added 0.051 grams of methylparaben and a trace of methylene blue dye. The mixture was heated to110° C., shaken and put on a vibro-mixer, and plunged into 23° C. waterfor 5 minutes. Two milliliters of a 2% Pluronic F-68 solution, acidifiedto pH 3 with HCl were overlaid, the test tube scaled with a twist cap,and the tube shaken and then sonicated for 30 minutes. This produced adispersion of microparticles coated with methylparaben.

Since this experiment alone with Example 10 demonstrate that particlescoated with the same compound, n this case methyl paraben, can beproduced either by a thermal method or by a chemical precipitationmethod this provides an extra degree of versatility which can beimportant in optimizing production efficiency and minimizing costs, forexample in large-scale pharmaceutical production of microencapsulateddrugs.

Example 9

The nanostructured reverse bicontinuous cubic phase material in thisexample is based on nonionic surfactants, which are generally approvedfor drug formulation, and which yield liquid crystalline phase materialswith properties tunable by small temperature changes. For example, in anacne cream this could be used to achieve detergent (cleansing)properties at the temperature of application, but insolubility at thetemperature of formulation. Furthermore, since it is based on a tunedmixture of two surfactants, and since the phase and properties thereofdepend sensitively on the ratio of the two surfactants, this provides aconvenient and powerful means to control the properties of the internalcore. In addition, this example resulted in a transparent dispersion.This is noteworthy because even a small fraction of particles with asize larger than about 0.5 microns gives rise to an opaque dispersion.

A nanostructured reverse bicontinuous cubic phase material was preparedby mixing 0.276 grams of “OE2” (an ethoxylated alcohol surfactantcommercially available as “Ameroxol OE-2”, supplied by Amerchol, adivision of CPC International, Inc.) with 0.238 grams of “OE5” (anethoxylated alcohol surfactant commercially available as “AmeroxolOE-2”, supplied by Amerchol, a division of CPC International, Inc.), andadding 0.250 grams of water (includes excess water). To this was added0.054 grams of methyl paraben and a trace of methylene blue dye. Themixture was heated to 110° C. shaken and put on a vibro-mixer, andplunged into 23 C water for 5 minutes. Two milliliters of a 2% PluronicF-68 solution, acidified to pH 3 with HCl, were overlaid, the test tubesealed with a twist cap, and the tube shaken and then sonicated for 30minutes. This produced a dispersion of microparticles coated withmethylparaben. Interestingly, the sub-micron size of the particlesresulted in a transparent dispersion.

Example 10

This example shows that methyl paraben-coated particles can be createdby a heating-cooling process, in addition to the acid-base method of theprevious example. This example also demonstrates that a mixture of twophases can be dispersed.

Lecithin (Epikuron 200, 0.418 grams) was mixed with 0.234 grams of oleylalcohol and 0.461 grams of acidic water at pH 3, resulting in a mixtureof nanostructured reverse bicontinuous cubic phase material andnanostructured reversed hexagonal phase material. Out of this was taken0.50 grams, to which were added 0.049 grams of methyl paraben, and mixedwell. This was heated to 120° C., stirred while hot, then reheated to120° C. The test tube was removed from the oven, and the test tubeplunged into cold water for 5 minutes. After this the twist-cap wastaken off, two milliliters of a 2% Pluronic F-68 solution, acidified topH 3 with HCl, were overlaid, and the sample stirred, shaken, andfinally sonicated. This resulted in a milky-white dispersion ofmicroparticles coated with methyl paraben. Examination in an opticalmicroscope showed microparticles with sizes in the range of 2–10microns. Excess methyl paraben crystalline material was also seen.

This example demonstrates that a mixture of two co-existingnanostructured phases can provide the interior of the microparticles.This could be important in, for example, controlled-release drugdelivery, where a mixture of two phases, each loaded with drug, could beused to achieve a desired pharmacokinetics: for example, with a mixtureof a reversed hexagonal phase and a cubic phase, the release from thesetwo phases follows different kinetics, due to the different geometry ofthe porespaces, and the resulting kinetics would be a combination ofthese two profiles.

Example 11

This example shows that water-free particle interiors can be produced,such as for protection of water-sensitive compounds.

The same procedure used in the preparation of Example 10 was used, butthe water was replaced by glycerol (which was present in excess) in thepreparation of the nanostructured bicontinuous reversed cubic phaseliquid crystalline material. The amounts were: lecithin 0.418 grams,oleyl alcohol 0.152 grams, glycerol 0.458 grams, and methyl paraben0.052 grams. The result was a milky-white dispersion of microparticlescoated with methyl paraben.

The protection of water-sensitive active compounds is important in, forexample, oral health care products incorporating actives that arehydrolytically unstable.

Example 12

In this example capsaicin was incorporated in particles coated withpotassium nitrate, and where the nanostructured reverse bicontinuouscubic phase material is based on extremely inexpensive surfactants. Thecoating is easily removed by simply adding water—such as in acrop-spraying gun which merges a stream of the dispersion with a streamof water, as it aerosolizes the liquid into droplets. Note that thepotassium nitrate would serve a dual purpose as a fertilizer.

The nonionic surfactants “OE2” (0.597 grams) and “OE5” (0.402 grams)were mixed with 0.624 grams of water which had been saturated withpotassium nitrate. To this mixture the active compound capsaicin (inpure crystalline form, obtained from Snyder Seed Corporation) was addedin the amount of 0.045 grams. Next, 0.552 grams of this mixture wereremoved, 0.135 grams of potassium nitrate were added, and the completemixture heated to 80° C. for 5 minutes. An upper solution was preparedby taking a 2% aqueous solution of Pluronic F-68 and saturating it withpotassium nitrate. The melted mixture was shaken to mix it, then putback in the 80° C. oven for 2 minutes. The test tube was the plunged in20° C. water for 5 minutes, at which point the upper solution wasoverlaid, and the entire mix stirred with a spatula, capped, shaken, andsonicated. The result was a dispersion of microparticles coated withpotassium nitrate, and containing the active ingredient capsaicin in theinterior.

When the dispersion was diluted with an equal volume of water, thecoating was dissolved (in accordance with the high solubility ofpotassium nitrate in water at room temperature), and this was manifestedas a rapid coagulation and fusion of the particles into large clumps.The interior of each particle was a tacky liquid crystal, so that in theabsence of a coating, flocculation and fusion occur.

The example that we discuss here is that of a spray that would be usedon decorative plants and/or agricultural crops, and would deter animalsfrom eating the leaves. We have succeeded in encapsulating the compoundcapsaicin, which is a non-toxic compound (found in red peppers andpaprika) that causes a burning sensation in the mouth at concentrationsin the range of a few parts per million. Capsaicin has a record ofcommercial use as a deterrent to rodents and other animals.

Pure capsaicin was encapsulated in the cubic phase interior of particleshaving a coating of crystalline potassium nitrate—saltpeter. Theexterior solution outside the particles was a saturated aqueous solutionof potassium nitrate, which prevents dissolution of the coating until itis diluted; a dilution of the dispersion with water by approximately 1:1resulted in nearly complete dissolution of the particle coatings. (Thisdissolution was captured on videotape and, on viewing the tape, it wasclear that there was dissolution of the coating and subsequent fusing ofthe particle interiors.)

Upon dilution and subsequent dissolution of the coating, the interior ofthe particle was exposed, this being a cubic phase with the followingcrucial properties:

A) it was insoluble in water;

B) it was extremely tacky, adhesive; and

C) it has very high viscosity.

Together these three properties imply that the de-coated cubic phaseparticles should adhere to plant leaves, and property A means that itwill not dissolve even when rained on.

The same three properties were also crucial to the success of animaltests of the bulk cubic phase, used as a controlled-release paste, inthe delivery of photodynamic therapy (PDT) pharmaceutical agents for thetreatment of oral cancer.

The concentration of capsaicin achieved in the cubic phase particles wastwo orders of magnitude higher than in pharmaceutical preparations usedin the treatment of arthritis. Higher loadings, perhaps as high as 20%may be possible.

From the standpoint of commercialization, the components in thedispersion are extremely inexpensive, and all are approved for use infoods, for topical application, and the like. In addition, potassiumnitrate is a well-known fertilizer.

Example 13

This example used capsaicin/potassium nitrate as in the previousexample, but here the nanostructured reverse bicontinuous cubic phasematerial is based on lecithin, which is an essential compound in plantand animal life, and can be obtained cheaply. This nanostructuredreverse bicontinuous cubic phase material is also stable over a widetemperature range, at least to 40° C. as might be encountered undernormal weather conditions.

Soy lecithin (Epikuron 200), in the amount of 1.150 grams, was mixedwith 0.300 grams of oleyl alcohol, 1.236 grams of glycerol, and 0.407grams of potassium nitrate. The active capsaicin was added to this inthe amount of 0.150 grams, and the mixture thoroughly mixed. Next, 0.50grams of potassium nitrate were added, and the complete mixture heatedto 120° C. for 5 minutes. An upper solution was prepared by taking a 2%aqueous solution of Pluronic F-68 and saturating it with potassiumnitrate. The melted mixture was stirred, then put back in the 120° C.oven for 3 minutes. The test tube was the plunged in cold water for 5minutes, at which point the upper solution was overlaid, and the entiremix stirred with a spatula, capped, shaken, and sonicated, thenalternated between shaking and sonicating for 30 cycles. The result wasa dispersion of microparticles coated with potassium nitrate, andcontaining the active ingredient capsaicin in the interior at a level ofapproximately 5%. Also present were crystals of excess potassiumnitrate.

The applications are similar to those of Example 12, except that the useof lecithin in the interior could provide for better integration of theparticle interior with the plant cell membranes, possibly yieldingbetter delivery.

Example 14

In this example cupric ferrocyanide-coated particles were shown to beresistant to shear.

A nanostructured reverse bicontinuous cubic phase material was preparedby mixing 0.296 grams of sunflower monoglycerides (Dimodan LS) with0.263 grams of a 10% aqueous solution of potassium ferrocyanide. Anupper solution was prepared by adding 0.021 grams of cupric sulfate and0.063 grams of Pluronic F-68 to 4.44 grams of water. The upper solutionwas overlaid onto the nanostructured reverse bicontinuous cubic phasematerial, the test tube sealed with a twist cap, and the systemsonicated for 45 minutes. The result was a high concentration ofmicroparticles, coated with cupric ferrocyanide, and with diameters onthe order of 3 microns. This process produces microparticles withoutrequiring temperature excursions, except those associated withsonicating, and these can be circumvented by using another form ofemulsification. Furthermore, no excursions in pH were required.

When a droplet was placed between microscope slide and cover slip formicroscopic examination, it was found that the cupricferrocyanide-coated particles were fairly resistant to shear; when thecover slip was massaged over the dispersion, light pressure with thefingers did not induce any noticeable loss of shape or fusion of theparticles. This was in contrast with, for example, particles coated withmagnesium carbonate hydroxide, where light pressure induced a highdegree of shape loss and fusing of particles. These observations were inaccordance with the high stiffness of cupric ferrocyanide.

Particles with coatings resistant to shear could be important inapplications requiring pumping of the particles, where traditionalpolymer-coated particles are known to suffer lifetime limitations due todegradation of the coating with shear.

Example 15

In this example capsaicin was incorporated at a fairly high loading,namely 9 wt %, into the interiors of crystal-coated particles of thepresent invention. A nanostructured reversed bicontinuous cubic phasewas produced by mixing 0.329 grams of lecithin, 0.109 grams oleylalcohol, 0.611 grams glycerol, and 0.105 grams of capsaicin (obtained incrystalline form as a gift from Snyder Seed Corp., Buffalo, N.Y.). Tothis cubic phase were added 0.046 grams of cupric sulfate. An uppersolution was prepared by mixing 0.563 grams of 10% potassiumferrocyanide aqueous solution with 2.54 grams of water. The uppersolution was overlaid onto the cubic phase-cupric sulfate mixture, andthe tube sonicated for two hours. The reaction that forms cupricferrocyanide was easily evidenced be the deep reddish-brown color of thecompound. At the end of this time, the cubic phase was dispersed intocupric ferrocyanide-coated particles. The coating was made of cupricferrocyanide, which is a strong material and has some selectivepermeability to sulfate ions. Since this coating material is a robustcrystal, as seen from Example 14, and capsaicin is extremely unpleasantto the taste of rodents, these particles could be useful as rodentdeterrents in preventing damage to corrugated boxes, agriculturalplants, etc., particularly where the particles must be resistant to mildshear (as during production of the particle-laced boxes, or depositionof the particles onto plants), prior to the gnawing action of rodentswhich would open the microparticles and expose the capsaicin to theanimal's tastebuds.

Example 16

In this example microparticles with a cupric ferrocyanide coating wereproduced using the same procedure as in Example 14, but in this case anantibody was incorporated as the active agent. In particular, anti 3′,5′ cyclic adenosine monophosphate (AMP) antibody was incorporated as anactive agent at a loading of 1 wt % of the interior. A cubic phase wasprepared by mixing 0.501 grams of sunflower monoglycerides with 0.523grams of water. Potassium ferrocyanide, in the amount of 0.048 grams,was added to the cubic phase, together with approximately 0.010 grams ofthe antibody. Excess aqueous solution was removed after centrifuging. Anupper solution was prepared by adding 0.032 grams of cupric nitrate and0.06 grams of Pluronic F-68 to 3.0 grams of water. After overlaying theupper solution and sonicating, a milky-white dispersion ofmicroparticles, coated with cupric ferrocyanide, was obtained. Suchparticles could be useful in a biotechnology setting such as abioreactor, in which the stiff cupric ferrocyanide coating would beuseful in limiting release during mild shear conditions encountered (forexample, in a pressurized inlet), prior to the desired release ofcoating and availability of the bioreactive antibody.

Example 17

In this example ethylhydrocupreine forms an extremely hard shell. Inthis example an acid-base process was used.

A nanostructured reverse biocontinuous cubic phase material was preparedby mixing 0.648 grams of sunflower monoglycerides (Dimodan LS) with0.704 grams of water. To this were added 0.084 grams ofethylhydrocupreine hydrochloride, and a trace of methylene blue An uppersolution was prepared by adding 1.01 grams of 0.1 M sodium hydroxide and0.052 grams of Pluronic F-68 to 3.0 grams of water. After overlaying theupper solution onto the liquid crystal, the system was sonicated,resulting in a dispersion of microparticles coated withethylhydrocupreine (free base). Most of the particles were less than amicron in size, when examined with optical microscopy.

Particles which maintain integrity with dessication could be useful in,for example, slow-release of agricultural actives (herbicides,pheromones, pesticides, etc.), where dry weather conditions could causepremature release of less resistant particles.

Example 18

In this example leucine-coated particles were created by aheating-cooling protocol.

A nanostructured reverse bicontinuous cubic phase material was preparedby mixing 1.51 grams of sunflower monoglycerides (Dimodan LS) with 0.723grams of water. To 0.52 grams of the nanostructured reverse bicontinuouscubic phase material taken from this mixture were added 0.048 grams ofDL-leucine. The mixture was stirred well and heated to 80° C., thencooled to room temperature by plunging in water. Immediately a 2%solution of Pluronic F-68 in water was overlaid, the mixture shaken, andthen sonicated. This resulted in a milky dispersion of microparticlescoated with leucine.

The ability to make the same coating (in this case leucine) by either athermal method or an acid-base method provides important flexibility inproduction, since, for example, certain actives (proteins, for example)are very easily denatured with temperature but can be quite resistant topH, whereas other compounds can be resistant to temperature but canhydrolyze at acidic or basic pH.

Example 19

This example shows that interior components can be protected fromcontact with oxygen even when oxygen was bubbled into the exteriormedium (here water).

A nanostructured reverse bicontinuous cubic phase material (with excesswater) was prepared by mixing 2.542 grams of sunflower monoglycerideswith 2.667 grams of water. From this, 0.60 grams of nanostructuredreverse bicontinuous cubic phase material were removed. Next, 0.037grams of DL-leucine and 0.497 grams of 1M HCl were mixed and dried,after which 0.102 grams of water were added, to yield a solution ofleucine hydrochloride, which was added to the 0.60 grams ofnanostructured reverse bicontinuous cubic phase material, along with atrace of methyl red dye. Then nanostructured reverse bicontinuous cubicphase material was a strong yellow color, but when spread out as a filmit turned crimson-red in about 3 minutes, due to oxidation. An uppersolution was prepared by mixing 0.511 grams of 1M sodium hydroxide,0.013 grams of Pluronic F-68, and 2.435 of water. A dispersion ofleucine-coated, methyl red-containing microparticles was prepared byoverlaying the upper solution onto the liquid crystal and sonicating. Itwas first checked that a solution of methyl red in water, with orwithout F-68 added, quickly changes from yellow to crimson-red when airwas bubbled through. Then, when air was bubbled through the dispersionof methyl red-containing microparticles, it was found that the color didnot change from yellow, thus demonstrating that the encapsulation of themethyl red inside the microparticles protected the methyl red againstoxidation.

Particles such as these which are able to protect the active compoundfrom contact with oxygen could be useful in protecting oxygen-sensitivecompounds, such as iron dietary supplements for example, during longstorage.

Example 20

In this example the water substitute glycerol was used both in theinterior nanostructured reverse bicontinuous cubic phase material, andas the exterior (continuous) coating, thus substantially excluding waterfrom the dispersion.

A dispersion of microparticles was prepared using glycerol instead ofwater, by mixing soy lecithin and oleyl alcohol in the ratio 2.4:1, thenadding excess glycerol and mixing and centrifuging. An amount of 0.70grams of this nanostructured reverse bicontinuous cubic phase materialwas mixed with 0.081 grams of methyl paraben. An upper solution wasprepared by adding cetylpyridinium bromide to glycerol at the level of2%. The nanostructured reverse bicontinuous cubic phase material-methylparaben mixture was sealed and heated to 120° C., mixed well, reheatedto 120° C., and then plunged into cold water, at which point the uppersolution was overlaid and the test tube re-sealed (with a twist-cap) andsonicated. This resulted in microparticles, coated with methyl paraben,in a glycerol continuous phase. Such a glycerol-based dispersion is ofinterest in the microencapsulation of water-sensitive actives.

Using microparticle dispersions such as these, hydrolytically unstableactives, which are encountered in a wide range of applications, can beprotected against contact with water even after release of the coating.

Example 21

Similar to Example 6 above, where zinc is used to challenge encapsulatedmethylene blue, but here the coating is potassium nitrate. In addition,the same dispersion is also subjected to challenge by potassiumdichromate.

A nanostructured reverse bicontinuous cubic phase material was preparedby mixing 0.667 grams of soy lecithin, 0.343 grams of oleyl alcohol,0.738 grams of glycerol, and a trace of methylene blue. To 0.469 gramsof the equilibrated phase was added 0.225 grams of potassium nitrate. Anupper solution was prepared by adding 2% Pluronic F-68 to a saturatedaqueous solution of potassium nitrate. This was overlaid onto the liquidcrystal, and the system sonicated until the liquid crystal was dispersedinto microparticles, coated with potassium nitrate. The color of thedispersion was light blue. Two tests were then used to show that themethylene blue was protected by encapsulation in the microparticles. Toapproximately 1 ml of this dispersion was added approximately 0.1 gramsof finely powdered zinc; when powdered zinc contacts methylene blue insolution, it causes a loss of color. After shaking, the mixture wascentrifuged very briefly, with about 10 seconds total time loading intothe centrifuge, centrifuging, and removing from the centrifuge, this wasdone to avoid interference from the zinc in determining the color of themethylene blue-containing particles. It was found that there was verylittle, if any, decrease in blue color from the treatment with zinc,showing that the microparticle coating protected the methylene blue fromcontact with the zinc. Then, potassium dichromate was added to anotheraliquot of the original light-blue dispersions. This changed the colorto a greenish color, with no hint of the purplish-brown that results ifmethylene blue in solution were contacted with potassium dichromate.

Coated particles of this Example feature an extremely cost-effectivecoating material, potassium nitrate, and yet protect active compoundsagainst chemical degradation from outside conditions, making them ofpotential importance in, for example, agricultural slow-release.

Example 22

This provides an example of microparticles with a permselective coatingof a inclusion compound. This particular inclusion compound, a so-calledWerner complex, has the property that the porosity remains when theguest molecule is removed. Clathrate and inclusion compound coatings areof interest as coatings of selective porosity, where selectivity forrelease or absorption can be based on molecular size, shape, and/orpolarity.

A nanostructured reverse bicontinuous cubic phase material was firstprepared by mixing 0.525 grams of sunflower monoglycerides and 0.400grams of water. To this were added 0.039 grams of manganese chloride(MnCl₂) and 0.032 grams of sodium thiocyanate. An upper solution wasprepared by adding 0.147 grams of 4-picoline (4-methylpyridine) to 3.0ml of a 2% aqueous solution of Pluronic F-68. The upper solution wasoverlaid on the liquid crystal mixture, and the test tube sealed andsonicated. The nanostructured reverse bicontinuous cubic phase materialwas thus dispersed into microparticles coated with the manganese form ofthe Werner complex, namely Mn(NCS)₂(4-MePy)₄.

The coating in this example may find use in the removal of heavy metalsfrom industrial streams. In this case the coating can be a porouscrystal—known as a clathrate—which permits atomic ions to pass acrossthe coating and into the cubic phase interior, which is an extremelyhigh-capacity absorbent for ions due to the high surface charge density(using an anionic surfactant, or more selective chelating groups such asbipyridinium groups, etc.). Most likely permanent pores would be thebest. The selectivity afforded by the clathrate coating circumvents thereduction in sorbent power that is inevitable with traditional sorbents(such as activated carbon and macroreticular polymers), due to largercompounds that compete with the target heavy metal ions for theavailable adsorption sites. Regeneration of the sorbent could be byion-exchange, while keeping the particles and coatings intact (thislatter step would, incidently, be an example of release).

Example 23

In this example coated particles with an outer coating comprising methylparaben and having a special dye disposed in the nanostructured reversebicontinuous cubic phase material were challenged with a cyanidecompound, which would cause a color change in the event of contact withthe dye. Since the cyanide ion is extremely small, the success of thistest shows that the coatings is impervious even to very small ions.

A nanostructured reverse bicontinuous cubic phase material was preparedby mixing 0.424 grams of sunflower monoglycerides and 0.272 grams ofwater. To this were added 0.061 grams of methyl paraben and a trace ofthe dye 1,2-pyridylazo-2-naphthol. An upper solution of 1%cetylpyridinium bromide was prepared. The liquid crystal was heated in a120° C. oven for five minutes, stirred vigorously, reheated, thenplunged into cold water, and which time the upper solution was overlaid,the test tube sealed, and put in a sonicator. The result was adispersion of methyl paraben-coated microparticles, with average size onthe order of 1 micron. Cuprous cyanide was then used to demonstrate thatthe dye was protected from contact with the exterior phase. When cuprouscyanide was added to a solution of 1,2-pyridylazo-2-naphthol (whether inthe presence of F-68, or not), the color changes from orange to strongpurple. However, when cuprous cyanide was added to an aliquot of thedispersion of dye-containing particles, there was no color change,showing that the dye was protected from contact with the cuprous cyanideby the methyl paraben coating. One can calculate that the diffusion timeof a cuprous ion into the center of a 1 micron particle is on the orderof a few seconds or less, which would not have prevented the colorchange had the coating not sealed off the particle.

The protection of active compounds from contact with ions from theoutside environment could be useful in, for example, drug delivery, inparticular in delivery of a polyelectrolyte which could be complexed andinactivated by contact with multivalent ions.

Example 24

In this example the cyanide ion test of the previous example wasrepeated for potassium nitrate-coated particles.

A nanostructured reverse bicontinuous cubic phase material was preparedby mixing 0.434 grams of sunflower monoglycerides and 0.215 grams ofwater. To this were added 0.158 grams of potassium nitrate and a traceof the dye 1,2-pyridylazo-2-naphthol. An upper solution of 1%cetylpyridinium bromide in saturated aqueous potassium nitrate wasprepared. The liquid crystal was heated in a 120° C. oven for fiveminutes, stirred vigorously, reheated, then plunged into cold water, atwhich time the upper solution was overlaid, the test tube sealed, andput in a sonicator. The result was a dispersion of potassiumnitrate-coated microparticles. When cuprous cyanide was added to analiquot of the dispersion of dye-containing particles, there was only aslight change of color, showing that the dye was substantially protectedfrom contact with the cuprous cyanide by the potassium nitrate coating.

The utility of these particles is similar to those in Example 23, butthe cost-effective coating potassium nitrate was used in this Example.

Example 25

A nanostructured reverse bicontinuous cubic phase material was preparedby mixing 0.913 grams of soy lecithin (Epikuron 200), 0.430 grams ofoleyl alcohol, and 0.90 grams of glycerol (excess glycerol). Aftermixing thoroughly and centrifuging, 0.50 grams of the nanostructuredreverse bicontinuous cubic phase material were removed and 0.050 gramsof dibasic sodium phosphate added. An upper solution was prepared byadding 0.10 grams of calcium chloride to 3 ml of an aqueous solutioncontaining 2% Pluronic F-68 and 1% cetylpyridinium bromide. Afteroverlaying the upper solution on the liquid crystal—sodium phosphatemixture, the test tube was sealed and sonicated. The result was adispersion of microparticles coated with a calcium phosphate. Calciumphosphate coatings were of inherent interest in biological contextssince calcium phosphates were a major component of bone, teeth, andother structural components.

Example 26

This example shows that the magnesium carbonate-coated particles in theexample retain their integrity upon dessication, that is, when theexterior water phase was dried off. Thus, dry powders can be producedwhile retaining the interior as a water-rich liquid crystalline phasematerial.

“Tung-sorbitol compound” preparation. Initially, a “tung-sorbitolcompound” was prepared as follows:

An amount of 110 grams of tung oil (obtained as Chinese Tung Oil fromAlnor Oil) was combined in a reaction flask with 11.50 grams ofsorbitol. The flask was purged with argon, sealed and heated to 170° C.and stirred magnetically. Sodium carbonate (3.6 grams) were added andthe mixture stirred at 170° C. for 1 hour. At this point, 3.4 grams of3-chloro-1,2-propanediol were added, and the mixture was cooled to roomtemperature. Seventy-five milliliters of the oily phase from thisreaction were mixed with 300 ml of acetone, and a white precipitateremoved after centrifugation. Next 18 grams of water and 100 ml ofacetone were added, the mixture centrifuged, and an oil residue on thebottom removed. Then 44 grams of water were added, and the bottom phaseagain collected and discarded. Finally, 20 grams of water were added andthis time the oily residue on the bottom collected and dried under argonflow. This yielded approximately 50 ml of a tung fatty acid ester ofsorbitol, which was referred to hereinafter as “tung-sorbitol product”.

Example 26A. A nanostructured reverse bicontinuous cubic phase materialwas prepared by mixing 0.110 grams of the “tung-sorbitol product”, 0.315grams of soy lecithin, and 0.248 grams of water, mixing thoroughly, andcentrifuging. To this was added 0.085 grams of potassium carbonate. Anupper solution was then prepared by adding 0.118 grams of Pluronic F-68and 0.147 grams of magnesium sulfate to 5.34 grams of water. The uppersolution was overlaid onto the liquid crystal, and the test tube sealed,shaken, sonicated for 2 hours, and finally shaken well again. The resultwas a milky-white dispersion of microparticles coated with magnesiumcarbonate hydroxide. This was diluted, by adding two parts water to onepart dispersion, in order to dissolve excess inorganic crystallinematerial. A small drop of the dispersion was spread gently onto thesurface of a microscope slide, and allowed to dry. After ten minutes ofdrying, the water exterior to the particles was almost completelyevaporated. Microscopic examination showed that the particlesnevertheless retained their shape, and did not become amorphous blobs,as was observed if uncoated particles were dried in a similar manner (asthe dried liquid crystalline mixture turns to a liquid).

Example 26B. The dispersion produced in Example 26A was heated to 40° C.According to phase behavior determinations, at this temperature theinterior phase was a nanostructured liquid L2 phase material. Thedispersion remained milky-white, and under the microscope showed theretention of microparticles as well. Since this L2 phase contains oil,water, and surfactant (namely the lecithin), it was also ananostructured microemulsion.

Example 27

In this example receptor proteins are disposed within the matrix of ananostructured reverse bicontinuous cubic phase material in the internalcore of magnesium carbonate-coated particles, then the coated particleswere in turn embedded in a hydrogel. The coating on the particles can beused to protect the receptor protein during shipping and storage, andthen easily removed by washing just before use. This example and Example28 presage the use of coated particles of the present invention for,e.g., affinity chromatography, using hydrogel beads with coatedparticles of the present invention embedded in them.

An amount of 0.470 grams of soy lecithin (Epikuron 200) was mixed with0.183 grams of the “tung-sorbitol product” (described above), and 0.359grams of water. To this was added 0.112 grams of potassium carbonate.This was centrifuged for several hours and the excess aqueous phaseremoved. A preparation of torpedo nicotinic acetylcholine receptor wasprepared according to the protocol described by L. Pradier and M. G.McNamee in Structure and Function of Membranes (ed. P. Yeagle, 1992, pp.1047–1106). In this preparation, 50 micrograms of receptor protein wascontained in 50 microliters of lipid, most of which wasdioleoylphosphatidylcholine (DOPC). (The remainder was other membranelipid components, such as other phospholipids, cholesterol, etc.) Thisamount of preparation was added to the nanostructured reversebicontinuous cubic phase material-potassium carbonate mixture, and theentire mixture stirred gently but long enough to ensure good mixing, aschecked by the absence of birefringence. An upper solution was preparedby adding 0.328 grams of magnesium sulfate, 0.324 grams of PluronicF-68, and 0.0722 grams of cetylpyridinium bromide to 20.02 grams ofwater. Five grams of the upper solution were overlaid onto the test tubecontaining the receptor-loaded nanostructured reverse bicontinuous cubicphase material, and the test tube sealed, shaken, and sonicated for 2hours. This resulted in a dispersion of magnesium carbonatehydroxide-coated, receptor-containing microparticles, a substantialfraction of which were in the size range of 0.5 to 1 micron.

The microparticles were then immobilized in a polyacrylamide hydrogel.Acrylamide (0.296 grams), methylene-bis-acrylamide (0.024 grams, ascrosslinker), ammonium persulfate (0.005 grams, as initiator), andtetramethylethylene diamine (TMED. 0.019 grams, as co-initiator) wereadded to the dispersion, resulting in polymerization of the acrylamideinto a crosslinked hydrogel in less than 30 minutes. A thin slice of thehydrogel was examined under a microscope, and a high concentration ofmicroparticles was seen, just as with the original dispersion.

The hydrogel was furthermore fragmented into bits with sizeapproximately 30 microns. This was accomplished by pressing the hydrogelthrough a wire mesh with a 40 micron mesh size.

Example 28

In this example receptor proteins were disposed in the internal core ofcoated particles of the present invention where the coating waspotassium nitrate, and the coated particles in turn immobilized inhydrogel beads. The receptor-laden beads were successfully tested forbinding activity in radioassays performed at UC Davis.

An amount of 0.470 grams of soy lecithin (Epikuron 200) was mixed with0.185 grams of the “tung-sorbitol product” (described above), and 0.368grams of water. To this was added 0.198 grams of potassium nitrate, andthe contents thoroughly mixed. A preparation of torpedo nicotinicacetylcholine receptor was prepared as described in the previousExample. In this preparation, per every 50 micrograms of receptorprotein was contained in 50 microliters of lipid, most of which wasdioleoylphosphatidyl-choline (DOPC). Fifty-five milligrams ofpreparation was added to the nanostructured reverse bicontinuous cubicphase material-potassium carbonate mixture, and the entire mixturestirred gently but long enough to ensure good mixing. An upper solutionwas prepared by adding 0.128 grams of Pluronic F-68 and 0.015 grams ofcetylpyridinium bromide to 6.05 grams of saturated aqueous potassiumnitrate solution. The nanostructured reverse bicontinuous cubic phasematerial-potassium nitrate preparation was heated to 40° C. to dissolvepotassium nitrate, then plunged into 10° C. water for 10 minutes. Theupper solution was overlaid onto the test tube containing thereceptor-loaded nanostructured reverse bicontinuous cubic phasematerial, and the test tube sealed, shaken, and sonicated for 2 hours.This resulted in a dispersion of potassium nitrate-coated,receptor-containing microparticles, a substantial fraction of which werein the size range of 0.3 to 1 micron.

The microparticles were then immobilized in a polyacrylamide hydrogel.Acrylamide (0.365 grams), methylene-bis-acrylamide (0.049 grams, ascrosslinker), ammonium persulfate (0.072 grams of a 2% solution, asinitiator), and tetramethylethylene diamine (TMED, 0.011 grams, asco-initiator) were added to the dispersion, resulting in polymerizationof the acrylamide into a crosslinked hydrogel in a matter of hours. Athin slice of the hydrogel was examined under a microscope, and a highconcentration of microparticles was seen (except near the very bottom ofthe hydrogel), just as with the original dispersion.

The hydrogel was furthermore fragmented into bits with sizeapproximately 30 microns. This was accomplished by pressing the hydrogelthrough a wire mesh with a 40 micron mesh size. At a 40 micron bit size,one can estimate that the diffusion time for a small molecule into thecenter of a bit is on the order of a second or less, which does not havea significant impact on the receptor tests reported next.

Using ¹²⁵I-labeled bungarotoxin as the ligand, an assay of receptorbinding was performed using the nanostructured reverse bicontinuouscubic phase material microparticle-immobilized acetylcholine receptorsystem just described. The standard assay for binding has been describedin publications from Dr. Mark McNamee's group. The results showed thatthe nanostructured reverse bicontinuous cubic phase materialmicroparticle-immobilized acetylcholine receptor system exhibitedbinding of the bungarotoxin at approximately 70% of the level measuredwith the standard receptor preparation, demonstrating the retention ofprotein binding properties throughout not only the immobilizationprocedure, but also the period (more than two months) that elapsedbetween the date on which the sample was prepared and the date it wastested.

In addition to demonstrating the production of particles of the instantinvention incorporating receptor proteins capable of targeting specificsites in the body, the foregoing Examples 26–28 actually show theapplication of the particles in biochemical assays, with a high degreeof improvement in stability over the commonly used liposomes, which areinconvenient due to their inherent instabilities. Such assays areimportant in clinical diagnoses, as well as in pharmaceutical drugscreening.

Example 29

As in Example 22 above, clathrate-coated particles were produced in thisexample. In this example the nanostructured reverse bicontinuous cubicphase material interior can be polymerized, by the effect of oxygenwhich can pass through the coating (the coating nevertheless preventspassage of water).

Lecithin extracted from Krill shrimp was obtained as Krill shrimpphosphatidylcholine from Avanti Polar Lipids of Birmingham, Ala. Anamount of 0.220 grams of this lecithin was mixed with 0.110 grams of“tung-sorbitol product”, 0.220 grams of water, 0.005 grams of a cobaltdryer (from the art materials supply company Grumbacher) containingcobalt naphthenate, and 0.30 grams of potassium thiocyanate. This formeda green-colored nanostructured reverse bicontinuous cubic phasematerial. An upper solution was prepared by adding 0.309 grams ofmanganese chloride, 0.105 grams of 4-picoline (4-methyl pyridine), 0.113grams of Pluronic F-68, and 0.021 grams of cetylpyridinium bromide to5.10 grams of water. The upper solution was overlaid on thenanostructured reverse bicontinuous cubic phase material, the test tubesealed, shaken, and sonicated, with ice water filling the sonicationbath water. As the green-color nanostructured reverse bicontinuous cubicphase material was dispersed into microparticles, the reaction caused acolor change to brown. After two hours, substantially all of thenanostructured reverse bicontinuous cubic phase material had beendispersed into particles, which were mostly submicron in size. Thecoating was a Werner compound, which according to the literature haschannels that allow the absorption of (or passage of) molecular oxygen.The high degree of unsaturation in the Krill lecithin, as well as thatin the tung-sorbitol product, together with the catalytic action of thecobalt dryer, makes it possible to polymerize this microencapsulatednanostructured reverse bicontinuous cubic phase material by contact withatmospheric oxygen.

The clathrates described in this example were discussed above (Example22).

Example 30

In this example a nanostructured reversed hexagonal phase material wasdispersed. A nanostructured reversed hexagonal phase material wasprepared by mixing 0.369 grams of soy lecithin (Epikuron 200), 0.110grams of sorbitan trioleate, and 0.370 grams of glycerol. To thisnanostructured reversed hexagonal phase material was added 0.054 gramsof magnesium sulfate. An upper solution was prepared by adding 0.10grams of potassium carbonate, 0.10 grams of Pluronic F-68, and 0.02grams of cetylpyridinium bromide to 5 grams of water. The upper solutionwas overlaid on the nanostructured reversed hexagonal phase material,and the test tube sealed, shaken and sonicated for one hour, resultingin a dispersion of most of the nanostructured reversed hexagonal phasematerial into microparticles coated with magnesium carbonate hydroxide.

The dimensionality of the pores (cylindrical) in the reversed hexagonalphase provides a unique release kinetics profile which could be usefulin, for example, controlled drug delivery.

Example 31

In contrast with most of the above examples, the nanostructured reversedhexagonal phase material that was dispersed in this example was not inequilibrium with excess water, although it was insoluble in water.

Soy lecithin (0.412 grams), linseed oil (0.159 grams), and glycerol(0.458 grams) were thoroughly mixed, producing a nanostructured reversedhexagonal phase material at room. temperature. To this nanostructuredreversed hexagonal phase material was added 0.059 grams of magnesiumsulfate. An upper solution was prepared by adding 0.10 grams ofpotassium carbonate, 0.10 grams of Pluronic F-68, and 0.02 grams ofcetylpyridinium bromide to 5 grams of water. The upper solution wasoverlaid on the nanostructured reversed hexagonal phase material, andthe test tube sealed, shaken and sonicated for 30 minutes, resulting ina dispersion of most of the nanostructured reversed hexagonal phasematerial into microparticles coated with magnesium carbonate hydroxide.

The ability to disperse nanostructured phases which are not inequilibrium with excess water expands the range of chemistries which canbe used in the present invention. This versatility is especiallyimportant in demanding applications, such as drug delivery, where alarge number of product criteria must be simultaneously satisfied.

Example 32

In this examples the nanostructured lamellar phase material wasdispersed using a chemical reaction process.

A nanostructured lamellar phase material was prepared by mixing 0.832grams of soy lecithin (Epikuron 200) and 0.666 grams of water. Toapproximately 0.80 grams of this nanostructured lamellar phase materialwas added 0.057 grams of magnesium sulfate. An upper solution wasprepared by adding 0.10 grams of potassium carbonate, 0.10 grams ofPluronic F-68, and 0.02 grams of cetylpyridinium bromide to 5 grams ofwater. The upper solution was overlaid on the nanostructured reversedhexagonal phase material, and the test tube sealed, shaken and sonicatedfor five minutes, resulting in a dispersion of most of thenanostructured lamellar phase material into microparticles coated withmagnesium carbonate hydroxide.

The particles in this Example bear a structural relationship withpolymer-encapsulated liposomes, but do not suffer from the harshchemical conditions used to produce polymer-encapsulated liposomes; theability to produce, in a single step, lamellar phase-interior particlescoated with a wide range of crystalline coatings, and under mildconditions, could make the present invention of importance in controlledrelease drug delivery.

Example 33

Preparation of free bases. Both ethylhydrocupreine and neutral red werepurchased in the protonated hydrochloride form. In each case this saltwas dissolved in water, to which was added aqueous sodium hydroxide in1:1 molar ratio. The mixture of the two aqueous solutions produced aprecipitate that was washed with water (to remove NaCl and any unreactedNaOH), centrifuged and then dried above the melting point of the freebase.

Preparation of nanostructured reverse bicontinuous cubic phasedispersions. Formulation of dispersions began with the followingmixture:

-   -   0.417 gm glycerol monooleate (GMO)    -   0.191 gm glycerol    -   0.044 gm ethylhydrocupreine (or, as the case may be, neutral        red, both in free base form).        Instead of the usual monoclyceride—water nanostructured reverse        bicontinuous cubic phase material, the monoglyceride—glycerol        nanostructured reverse bicontinuous cubic phase material was        used in these examples.

An upper solution was made by dissolving Pluronic F-68 in water to alevel of 2%.

After weighing the components into a test tube and mixing with aspatula, the sealed (twist-cap) test tube was put in a 140° C. oven forat least 20 minutes, and the ethylhydrocupreine (or neutral red freebase) was checked to have melted. The test tube was then plunged intowater, which was below room temperature (about 10° C.) in some cases androom temperature water in others: no difference was found in thedispersions in the two cases.

After the sample had been in the cooling water for about 5 minutes, theviscosity was checked to be very high, indicating a nanostructuredreverse bicontinuous cubic phase: in some cases the sample was observedthrough crossed polars for optical isotropy (the crystalline coatingdomains are mush smaller than the wavelength of light, too small toaffect the optical properties). The Pluronic upper solution was pouredinto the test tube until about half full. The tube was then shaken, byhand and with the use of a mechanical mixer. The solution becameincreasingly opaque as the bulk nanostructured reverse bicontinuouscubic phase material disappeared and went into dispersion.

SEM characterization. Scanning electron microscope (SEM) preparation didnot involve any fixation technique whatsoever. A drop of dispersion wassimply placed on a glass slide, the water evaporated, and a thin (2 nm)coating of carbon sputtered on to avoid charging effects. In thesputtering apparatus, before sputtering began the sample wasdeliberately held for about 5 minutes at a vacuum of 5×10⁻⁴ Torr. Thiswas done to test the robustness of the particle coating. The SEM usedwas a Hitachi S-800 field-emission SEM, and was operated at 25 kV.

FIG. 3 shows an SEM micrograph of an ethylhydrocupreine dispersion, andparticles in the range of about 0.5–2 micron diameter are seen (thebottom half is a 10× magnification of the area boxed in the top half, sothat the magnification is 500 on top and 5,000 on the bottom). Many ofthe particles, remarkably, distinctly show a polyhedral shape.

The measured particle size distribution for this sample (see the nextsection) showed that particles on the order of 0.5–2 microns diameterdominate in this dispersion, and this agrees well with the particlesseen in the micrograph. One can estimate that the thickness of theethylhydrocupreine coating in a 0.5 micron particle was about 10 nm, andthis was clearly thick enough that it was able to protect the liquidcomponents in the interior of the particles from evaporation in the 0.5mTorr vacuum.

In this dispersion, the nanostructured reverse bicontinuous cubic phasematerial was loaded with lithium sulphate as a marker before dispersing,and indeed the EDX spectra of particles in this dispersion showed asulfur peak. Lithium cannot be detected by the EDX used, and other peaksin the spectrum were attributed to the glass substrate.

FIG. 4 shows an SEM micrograph of a neutral red dispersion.Substantially all of the particles have sizes in the range of 0.3–1micron.

Particle size distribution. A Malvern 3600E laser diffraction particlesizer was used to measure the distribution. For each dispersion checked,a few drops were added to the carrier fluid (water), resulting in alarge dilution of the concentration so as to avoid multiple scattering.The particle size was computed as the diameter of a sphere of the samevolume, which is a good measure considering the polyhedral shape of theparticles. (See below.) The instrument is capable of measuring particlesdown to at least 0.5 micron, and data on the distribution includecontributions at least down to 0.5 microns.

The particle size distribution of a dispersion prepared with a 13:1ratio of GMO:ethylhydrocupreine is shown in FIG. 5. In general, as theratio of nanostructured reverse bicontinuous cubic phase material tocrystalline coating agent increases so does the particle size. The datafor this dispersion show that, on a volume average basis, 10% of theparticles have particle size less than 0.6 microns this beingrepresented by the equation D(v,0.1)=0.6 micron. The narrowness of thedistribution is indicated in two ways. First, the D(v,0.9) and D(v,0.1)are each a factor of 2 from the (volume-weighted) average ofD(v,0.5)=1.2 micron. And second, the “span”, which gives the width ofthe distribution as:span=[D(v,0.9)−D(v,0.1)]/D(v,0.5)is computed to be 1.4. These results indicate a fairly low degree ofagglomeration.

A narrower distribution was indicated for a dispersion with GMO:neutralred=10:1. The span is given as 1.1, and the (differential) particle sizedistribution was easily seen to be (quite sharp, dropping off quicklyabove 2 micron.

A small particle size was measured for a dispersion prepared with alower GMO:ethylhydrocupreine ratio, with a distribution averaging 0.8microns, and a span of 1.2. Thus, particle size can be controlled by theratio of nanostructured reverse bicontinuous cubic phase material tocrystalline coating agent, with the particle size decreasing withdecreasing ratio.

Small-angle X-ray scattering (SAXS). This was used to verify that theinterior of the particles in an ethylhydrocupreine dispersion was ananostructured reverse bicontinuous cubic phase material. The dispersionitself—not a concentrate of the particles—was loaded into a 1.5 mm x-raycapillary, which was transported to the laboratory of Dr. Stephen Hui atRoswell Park Cancer Center Biophysics Department. The SAXS camera wasequipped with a rotating anode, and measurements were performed at 100kV, 40 mV power (4 kW). Data were collected using a linearposition-sensitive detector connected through an electronics setup to aNucleus multichannel analyzer. The MCA has the capacity for 8,192channels, but only 2,048 resolution was used to increase the counts perchannel. Counting times on the order of an hour were used because thevolume fraction of nanostructured reverse bicontinuous cubic phasematerial in the dispersion (which was about 85% of the particle volume)was on the order of 10%. The software package “PCA” was used foranalysis of the data.

FIG. 6 shows the measured SAXS intensity versus wave vector q plot. Thewave vector q is related to the diffraction angle θ and the wavelength λof x-rays by the formula:q=4π(sin θ)/λ.A d-spacing is calculated from the q-value of a Bragg reflection by:d=2π/q.

In FIG. 6, vertical lines given the exact, calculated Bragg peakpositions for a lattice with space group Pn3m and lattice parameter 7.47nm. This space group is well-established for nanostructured reversecubic phases in the monoolein-water system, particularly for those whichare in equilibrium with excess water. (Indeed, in nanostructured reversecubic phases which are in equilibrium with excess water, the space groupPn3m almost exclusively appears). Lattice parameters for themonoolein-water nanostructured reverse cubic phase with space group Pn3mare also close to 8 nm; a more exact comparison was impossible becauseof the substitution of glycerol for water in the present case. In anycase, the lattice type and size deduced from this SAXS scan are in exactaccord with literature data for monoglyceride nanostructured reversecubic phases.

In the space group Pn3m, the Miller indices (hkl) for the allowed peakpositions, and the value of h²+k²+l², are: (110), 2; (111), 3; (200), 4;(211), 6; (220), 8; (221), 9; (222), 12; and higher. Looking at the dataand the expected peak positions, it is clear that the peaks at the (110)and (222) positions are strongly supported by the data. The (111) peakappears as a shoulder to the (110) peak on the right side of the scan,and as a small but discernible peak on the left side. The (200) peak issupported at least on the right side of the scan: this peak is alwaysmeasured to be much less intense than the (110) and (111) peaks inmonoglyceride Pn3m phases, and in Pn3m phases in general, and this hasbeen found to be in accord with theoretical amplitude calculations[Strom, P. and Anderson, D. M. (1992) Langmuir, 8:691]. The (211) peakis supported by data on the left side of the scan, and the (221) by dataon the right side. The absence or low intensity of peaks between the(211) and (222) is a consequence of the low concentration (10%) ofnanostructured reverse bicontinuous cubic phase in the dispersion, sincethe intensity of diffracted x-rays varies as the square of the volumeconcentration. Despite this, the definitive peaks at the (110) and (222)positions, and the perfect agreement of the deduced lattice and latticeparameter with related systems in the literature provide strong supportfor the conclusion that the SAXS data demonstrate nanostructured reversebicontinuous cubic phase ordering in the particle interiors.

These particles could be useful in, for example, controlled release ofantiseptics in oral rinses, where the solubilities of the two coatingsat slightly lowered pH (on the order of 5) was in the right range tomake delivery preferential at sites of bacterial activity.

Example 34

High-performance liquid chromatography (HPLC) was used to characterizethe integrity under shear and pressure of two dispersions, one chosen tohave a rigid coating—cupric ferrocyanide—and the other a soft, easilydisrupted coating, the latter, to act essentially as a control, toquantity any release under pressure of the more rigid coating. In otherwords, if the concentration of marker in the two dispersions wereapproximately the same, and the release of marker in the rigid systemwere a small fraction, say, x % (where x is substantially less than100), of the release of marker in the soft system, then one couldconclude that only x % of the particles in the rigid system broke upunder the pressure, and the remaining (100−x) % remained intact duringthe HPLC. Indeed, this percentage 100−x is a lower limit; the actualpercentage of intact rigid particles would be calculated to be higher ifit were found that some fraction of the soft particles in the controlhad actually remained intact, though this possibility is remote. In anycase, the calculations were assumed to be on a worst case scenario, byassuming that all the control particles broke up.

Preparation of the Dispersions

Example 34A. A nanostructured reverse bicontinuous cubic phase materialwas prepared by mixing 0.499 grams of soy lecithin, 0.163 grams of oleylalcohol, 0.900 grams of glycerol, and 0.124 grams of capsaicin. To 0.842grams of the nanostructured reverse bicontinuous cubic phase materialfrom this system was added 0.043 grams of sodium cholate. An uppersolution was prepared by adding 1 drop of 1M HCl to 3.00 grams of pH 5phosphate buffer. The upper solution was overlaid onto the liquidcrystalline material, and the test tube sealed and sonicated, resultingin a milky-white dispersion of microparticles.

Example 34B. A nanostructured reverse bicontinuous cubic phase materialwas prepared by mixing 0.329 grams of soy lecithin, 0.108 grams of oleylalcohol, 0.611 grams of glycerol, and 0.105 grams of capsaicin. To thiswere added 0.046 grams of cupric sulfate. An upper solution was preparedby adding 0.563 grams of 10% potassium ferrocyanide solution to 2.54grams of water. The upper solution was overlaid onto the liquid crystaland the test tube sealed and sonicated, resulting in a milky-whitedispersion of microparticles coated with cupric ferrocyanide.

The concentration of marker, namely capsaicin, was comparable in the twosamples. The final concentration in the cupric ferrocyanide dispersionwas 2.44%, compared to 3.19% for Example 34B—a 30% difference, whichwill be accounted for in the calculations below.

Purified capsaicin was then run in HPLC and found to have all elutiontime of 22 minutes (data not shown). Under these identical conditions,the two dispersions prepared above were run. The data for the particlesof Example 34B is shown in FIG. 7, and for the cupric ferrocyanideparticles in FIG. 8. Tables 1 and 2 give the integrated peakscorresponding to FIGS. 7 and 8, respectively, as output from the HPLCcomputer; sampling rate was 5 Hz.

Clearly there is a strong peak in FIG. 7 at 22 minutes elution time(numbered peak 13 by the computer), and Table 1 gives the integratedintensity of this peak as 3,939,401. A much smaller peak is seen at 22minutes in FIG. 8 (numbered 10 by the computer), and Table 2 gives theintensity as 304.29.

If these integrated peak values are normalized according to theconcentration of capsaicin in the two samples, namely 3,939,401/0.0319for the Example 34B case and 304,929/0.0244 for the cupric ferrocyanidecase, the ratio of the normalized peak intensity for the cupricferrocyanide case to the Example 34B case is 0.101—that is, at most10.1% of the cupric ferrocyanide particles released the capsaicin markerunder the HPLC conditions.

These particles have a coating which is a mineral of low aqueoussolubility, making them of potential utility in applications requiringrelease of the particle coating by strong shear, while at the same timeprotecting against release due to simple dilution with water. An exampleof such an application would be where a rodent deterrent such ascapsaicin, or rodent toxin, would be encapsulated, the particlesimpregnated into electrical wires, corrugated boxes, and other productsrequiring protection against gnawing by rodents, and the gnawing actionof a rodent would induce release of the active deterrent or toxin. Thelow water solubility would prevent the deterrent from premature releasedue to damp conditions.

TABLE 1 Integrated peak intensities corresponding to FIG. 7 for HPLCanalysis of Example 34B particles containing capsaicin. Peak #13 is themain capsaicin peak. Peak Area 1 2914 2 8096 3 2848 4 29466 5 11304 62254 7 12871 8 4955 9 124833 10 113828 11 19334 12 7302 13 3939401 1439153 15 255278 16 755868 17 52623 18 19395 19 4899 20 10519 21 5101 221481 23 344230 24 9971 25 194442 26 89831 27 80603 28 105163 29 18622430 194020 31 36805 32 2115 33 23296 34 4327 35 5166 36 90236 37 62606 3844523 39 110347 40 4391 41 1275597 42 1353000 43 238187

TABLE 2 Integrated peak intensities corresponding to FIG. 8 for HPLCanalysis of cupric ferrocyanide-coated particles containing capsaicin.Peak #10 is the main capsaicin peak. Peak Asea 1 1681172 2 3011240 3106006 4 2760 5 59059 6 38727 7 163539 8 44134 9 6757 10 304929 11 1046612 141800 13 332742 14 14442 15 6996 16 15008 17 11940 19 91446 20250214 21 251902 22 203000 23 44658 24 110901 25 24296 26 19633 27 2552728 15593 29 75442 30 40245 31 421437

Example 35

A nanostructured cubic phase liquid crystal was prepared by mixing 0.77grams of soy lecithin (Epikuron 200, from Lucas-Meyer), 0.285 grams ofoleyl alcohol, and 0.84 grams of glycerol, to which was added 0.11 gramsof auric chloride. No heating was used in the equilibrium of thismixture, only mechanical stirring with a spatula. An amount 0.595 gramsof this mixture was removed and smeared along the bottom half of theinner surface of a test tube. An upper solution was prepared bydissolving 0.14 grams of ferrous chloride and 0.04 grams of PluronicF-68 in 1.74 grams of distilled water. After overlaying the uppersolution the test tube containing the cubic phase was sonicated,resulting in a dispersion of microparticles coated with a gold coating.A control sample, in which the upper solution contained the F-68 but noferrous chloride, was sonicated side by side with the first sample anddid not result in a dispersion of microparticles. The reaction betweenferrous chloride and auric chloride results in the precipitation ofelemental, nonlamellar crystalline gold, which in the case of the firstsample resulted in the creation of microparticles covered with gold,with cubic phase interior. A glycerol-water mixture with a densityapproximately 1.2 gram/cc was then prepared by mixing 0.62 g of glycerolwith 0.205 grams of water, and approximately 0.1 grams of the dispersionwas added to this, and the new dispersion centrifuged. A substantialfraction of the microparticles could be centrifuged to the bottom ofthis test tube after centrifuging for 3 hours, demonstrating that thedensity of these particles was significantly higher than 1.2; this wasdue to the presence of the gold coating, since the density of the cubicphase was less than 1.2—indeed, a portion of the cubic phase which wasnot dispersed during the time of sonication could be centrifuged out ofthe original dispersions as a lower-density band, showing that thisliquid was even less dense than the original dispersion.

Because gold is well-known for exhibiting chemical inertness—as well asgood mechanical properties when in the form of very thin films, andsince it is also approved by the FDA for many routes of administration,gold-coated particles could be useful in safe, environmentally-friendlyproducts demanding chemically and physically stable coatings.Furthermore, such particles could be effective in the treatment ofarthritis, by providing greatly increased surface area of gold overother colloidal forms.

Example 36

A nanostructured liquid phase containing the antineoplastic drugPaclitaxel was prepared by solubilizing 0.045 grams of Paclitaxel, 0.57grams of eugenol, 15 grams of soy lecithin (Epikuron 200), 0.33 grams ofglycerol, and 0.06 grams of cupric nitrate with 0.61 grams of methanol,and then evaporating off the methanol in an evaporating dish, withstirring during evaporation. An glycerol-rich upper solution wasprepared by dissolving 0.09 grams of potassium iodide, 0.05 grams ofPluronic F-68, 0.44 grams of water and 1.96 grams of glycerol. Afteroverlaying the upper phase, the system was sonicated, resulting in thedispersing of Paclitaxel-containing, nanostructured liquid phase intomicroparticles coated with crystalline iodine. Since these ingredientswere chosen for their general acceptance as safe, inactive (except forthe Paclitaxel itself) excipients in pharmaceutical preparations, thisformulation or a variation thereof could be of importance in thedelivery of Paclitaxel for the treatment of cancer. The loading ofPaclitaxel in the particle interior was quite high, namely on the orderof 3 wt %, which in this case was so high that precipitation of some ofthe Paclitaxel within the interior of each particle may occur since thesolubilization of Paclitaxel in this cubic phase at this high loadingwas metastable. However, studies indicate that the precipitation is veryslow, taking hours or even days, at such loadings, so that substantiallyall of the Paclitaxel remains in solution during the course of theproduction of particles; thereafter, the confinement of the Paclitaxelwithin the coated particles prevents the formation of large crystals(larger than a micron). If the concentration of Paclitaxel in thissystem were lowered, to 0.7% or less of the interior, then thesolubilization of Paclitaxel becomes a truly stable solubilization(thermodynamic equilibrium) so that precipitation is preventedaltogether, and microparticles of the present invention coated withnonlamellar crystalline iodine can be produced as described in thisExample. Thus this system provides several scenarios for use inPaclitaxel delivery for cancer treatment.

Example 37

A Paclitaxel-containing cubic phase liquid crystal was prepared bymixing 0.345 grams of soy lecithin (Epikuron 200), 0.357 grams ofanisole, 0.26 grams of water and 0.02 grams of Paclitaxel (from LKTLaboratories); equilibration was speeded by plunging a test tube of themixture, after vigorous stirring, into boiling water for one minute thencooling to room temperature. To provide a coating material, 0.07 gramsof propyl gallate was stirred in and the test tube again heated inboiling water. It had previously been checked that propyl gallate doesnot dissolve appreciably in this cubic phase at room temperature, butthat the solubility increases substantially at 100° C. An upper solutionconsisted of 2.25 grams of a 2% Pluronic F-68 solution. The cubicphase-propyl gallate mixture was heated to 100° C. cooled to about 80°C. stirred with a spatula at the elevated temperature, and reheated to100° C. After cooling the mixture for about 30 seconds, the uppersolution was then overlaid on this mixture and the test tube placed in asonication bath for one hour. A dispersion of microparticles with aPaclitaxelcontaining interior and coated with propyl gallate wasobtained. The dispersion had a high concentration of extremely finemicroparticles (estimated particle diameter less than 0.4 micron), whichwere observable in the optical microscope at 1000× by virtue of theirBrownian motion. The overall particle size distribution was fairlybroad, with some particles as large as 1–2 microns. Only a very smallamount of precipitated Paclitaxel, in the form of needles, was observed,so that nearly all of it must be in the interiors of the microparticles.The concentration of Paclitaxel in this example was high enough that thesolubilization was metastable, which has implication as discussed in theprevious example. Since the concentration of the antineoplastic drugPaclitaxel in the interiors of these particles was about 2%, and thecomponents of the formulation are on the FDA list of approved inactiveexcipients for oral delivery, (and nearly all of them for injection aswell), this formulation could be very important as a drug-delivery,formulation for the treatment of cancers.

Example 38

The amphiphilic polyethyleneoxide-polypropyleneoxide block copolymerPluronic F-68 (also called Poloxamer 188), in the amount of 1.655 grams,was mixed with 0.705 grams of eugenol and 2.06 grams of water. Uponcentrifugation, two phases resulted, the bottom phase being ananostructured liquid phase, and the top a nanostructured cubic phase.An amount of 0.68 grams of the liquid crystalline phase was removed, andto it were added 0.05 grams of sodium iodide. A drop of eugenol wasadded to 2.48 grams of the lower phase to ensure low viscosity, and thisnanostructured liquid phase, with 0.14 grams of silver nitrate added,served as the “upper solution” in dispersing the liquid crystallinephase. Thus, the liquid phase was overlaid on the liquid crystallinephase containing the iodide, and the mixture sonicated for 1.5 hours.The result was a dispersion of silver iodide-coated particles in anexternal medium of the nanostructured liquid phase.

This Example illustrates the use of nanostructured liquid crystallinephases based on block copolymers as interior matrices for particles ofthe present invention. In this case, water was used as a preferentialsolvent for the polyethyleneoxide blocks of the block copolymer, andeugenol as preferential solvent for the polypropyleneoxide blocks of theblock copolymer (which are insoluble in water).

This Example also illustrates the use of a general approach discussedabove, namely the use of a nanostructured phase as the mixture thatserves as the “upper solution”, providing moiety B which reacts withmoiety A in the interior phase to cause precipitation of a crystallinecoating material. In this case, B is the silver nitrate, which inducesprecipitation of silver iodide on contact with the interior matrix A(the cubic phase) which contains sodium iodide. As discussed above, itis generally desirable to choose this upper solution so that it is inequilibrium with the interior matrix, or, as in this case, very nearlyso (the only deviation from true equilibrium being due to the additionof a single drop, about 0.01 grams or less than 0.5% of eugenol to theupper solution). As in this approach, is generally useful to choose theinterior matrix so that it is a viscous material, much more so than theupper solution which should be of relatively low viscosity.

Example 39

A poly(lactic-glycolic acid) polymer (PLGA), with a 59:41lactide:glycolide ratio and an inherent viscosity of 0.51 dl/gm, wasobtained from Purac Biochem (The Netherlands). This copolymer is knownto be amorphous, and this was evidenced by lack of birefringence. Anamount 0.307 grams of this polymer was dissolved in 3.002 gm of ethylacetate. A cubic phase was prepared by mixing 0.042 grams of theprothrombogenic compound menadione, 0.272 grams of oil of ginger, 0.224grams of water, and 0.540 grams of the ethoxylated hydrogenated castoroil surfactant Arlatone G (obtained from Uniquema). This was heated to50° C. in order to dissolve the menadione. An amount 0.302 grams of thiscubic phase was added to a second 16 ml glass tube, overlaid with 9.707ml of water, and dispersed into the water by shaking. The PLGA solutionwas added to the cubic phase dispersion, the mixture shaken immediately,and sonicated for 10 minutes. Following this, the contents weretransferred into a round bottom flask, placed on a rotovap apparatus,and evaporated to a final volume of approximately 9.7 ml.

This resulted in PLGA-coated particles of two types. First, in the waterphase, comprising approximately 2% by volume, were microparticles ofcubic phase coated with PLGA. A significant fraction of thesemicroparticles were large enough to see structural detail in aphase-contrast optical microscope. An optical micrograph is shown inFIG. 9. The shell is visible in the larger particles. The irregularthickness of this shell layer is evidence that this layer is not anoptical artifact. This is also evident when adjusting the focus on themicroscope: if this were an artifact, its thickness would change as thefocus changed, and this does not occur.

The second type of particle that came out of the process was a large,millimeter-sized particle that clearly behaved as a solid-coatedparticle. In one experiment, a reddish-orange dye, methyl red, which isof low, solubility in both water and ethyl acetate, was dissolved in thecubic phase prior to dispersing. In addition to a reddish-orange tingeto the microparticles seen in the microscope, the millimeter-sizedparticles were strongly red-orange, demonstrating that the cubic phaseis encapsulated inside the PLGA. Millimeter-sized particles of this typecould be suspended on the tip of a needle, for example, without flowing,in contrast with uncoated cubic phase which could not be suspended inthis fashion.

One of these large particles was placed in linalool, which is a solventfor the cubic phase but not for the PLGA. The particle did not dissolvein this solvent even after one week, whereas the cubic phase withoutPLGA coating dissolved in less than 5 minutes. FIG. 10 shows aside-by-side comparison of the PLGA-coated (on the left) and uncoated(on the right) cubic phases soaking in linalool, demonstrating clearlythe insolubility of the coated cubic phase—the original color photographshows that there is essentially no color to the linalool for thePLGA-coated sample, whereas the linalool containing the uncoated cubicphase is strongly red-orange. This experiment proves that the cubicphase is truly encapsulated by the PLGA.

Example 40

A cubic phase containing solubilized methyl red was first prepared bymixing 2.118 grams of Arlatone G, 0.904 grams of water, 1.064 grams ofoil of ginger, and 0.012 grams of methyl red, and stirring thoroughly. Atrehalose solution was prepared by dissolving 2.00 grams of trehalose in10.005 grams of water. Then 1.002 grams of the cubic phase weredispersed in the trehalose solution by a combination of shaking and mildsonication. This dispersion was then freeze-dried in a lyophilizer.Trehalose solutions are known to yield amorphous solid on freeze-drying.The resulting material flowed freely, and gave no hint of the greasy,sticky feel and behavior for that characterizes the uncoated cubicphase. There was no second phase present, as the material washomogeneous to the eye, and had a strong, uniform, red-orange color. Alarge particle of the material was speared with the point of a push-pinand photographed, as shown in FIG. 11; an uncoated cubic phase would nothave been possible to spear and suspend indefinitely in this fashion.

In the phase-contrast optical microscope, thin portions of this materialwere readily seen to contain a fine-scale structure, which is consistentwith the presence of cubic phase microparticles (submicron to 5 micronsin size) within the trehalose solid matrix. The material was brittle andcould therefore be crushed into small particles with ease. Upon mixingthe material into water at, say, a 1:10 ratio, a dispersion wasimmediately obtained which was indistinguishable in the opticalmicroscope from dispersions of this cubic phase in water.

Example 41

This Example demonstrates a method of production of coatedmicroparticles in which a precursor to the coating material, which issurface-active when dissolved in water, is used to disperse a cubicphase into particles; then after reacting to convert this precursor to asolid coating, energy input is again applied to reduce the particle sizeto submicron. As discussed above, one advantage of this method is thatit localizes the coating precursor at the particle surface, so that thecubic phase readily becomes encapsulated upon conversion of thisprecursor to the coating. The active compound in this Example wastriclosan.

A cubic phase was prepared by mixing 0.886 grams of linalool, 0.960grams of Pluronic P123 (BASF), 0.104 grams of triclosan, 0.189 grams of2-ethylhexanoic acid, and 0.879 grams of distilled water, and thenstirring thoroughly. This cubic phase was then smeared onto the sides ofa test tube, 3.33 grams of a sodium N-acetyltryptophan (Na-NAT) solution(6 wt % based on the NAT) overlain, and the mixture shaken and sonicatedbriefly to disperse the cubic phase; the Na-NAT thus acts as adispersant or surfactant in this step. A 30% zinc acetate solution, inthe amount 0.37 grams, was then added and mixed with the dispersion,followed by 0.52 grams of 2N NaOH. Five minutes were allowed for thereaction to begin, after which the material was further sonicated. Asurfactant solution (0.10 grams) containing Cremophor EL (9%) andPluronic F-68 (12.5%) was then added, and the mixture sonicated for 15minutes. The solid-coated nature of the resulting microparticles wasevident in phase contrast optical microscopy, where shearing thedispersion between glass and coverslip clearly showed that themicroparticles behaved as solid-coated particles rather than as thereadily-deformable cubic phase particles that result without applicationof the coating.

Example 42

This Example reports a process in which coating material is melted, anda cubic phase dispersed therein, following which the temperature islowered to solidify the coating, after which energy input is applied tocreate particles. Such a process can be applied to crystalline materialsas well as to amorphous or semi-crystalline coating materials, where inthe case of an amorphous material the cooling may result in an amorphousmaterial (and is thus not a true “freezing”, but rather avitrification).

The nutriceutical compound Coenzyme Q10 was incorporated into a cubicphase based on the ethoxylated, hydrogenated castor oil surfactantArlatone G (from Uniquema). Coenzyme Q10 (10 mg) was solubilized in amixture of 0.302 grams of essential oil of ginger, 0.201 grams of water,and 0.606 grams of Arlatone G. This cubic phase was placed in a testtube and 2.994 grams of hydrogenated cottonseed oil added, and theentire contents were heated to 90° C. to melt the oil. The sample wasimmediately sonicated in a hot water bath with vigorous shaking every 30seconds, for 3 minutes. The test tube was then placed in an ice bath tosolidify the oil with particles dispersed throughout the trigylceride.The resulting solid was then milled by the application of mechanicalenergy to an average particle size of several hundred microns; furtherreduction in size can readily be accomplished by milling methods wellknown in the art.

Example 43

This Example shows that a lectin incorporated into a cubic phasemicroparticle—the microparticle that would result after the dissolutionof the zinc N-acetyltryptophan coating of a particle of the typeproduced in Example 41—retains its ability to bind oligosaccharides.

A cubic phase was first prepared by mixing 0.752 grams of Pluronic P123(an insoluble surfactant), 0.705 grams of linalool, and 0.703 grams ofwater. An amount of 1.005 grams of this cubic phase was put in a glassflask together with 0.054 grams of the rhamnolipid surfactant JBR-99(Jeneil Biosurfactant, Inc.) and 35 ml of pH 4.5 acetate buffercontaining 4 nM, MnCl₂ and 4 mM CaCl₂. The flask was then sonicated todisperse the cubic phase. Following this, the dispersion wasmicrofluidized in a model 110S Microfluidizer (Microfluidics, Inc.) to aparticle size that was fine enough where the absorbance measured on anUltrospec 3000 UV-Vis spectrometer, at a wavelength of 620 nm, was about0.2 absorbance units.

The following reagents were then added to 2 ml of the cubic phasedispersion: Anti Concanavalin A, Vector AS-2004, Lot 0321, 1 mg/ml stocksolution prepared: working solution prepared by diluting 1:10 to 0.1mg/ml: 51 microliters added. Concanavalin A, Sigma C-5275, Lot 60K8934prepared as 1 mg/ml stock solution; working solution prepared bydiluting 1:10 to 0.1 mg/ml: 16 microliters added. Biotinylatedmannotriose, V-labs, NGB1336, prepare a 1 mg/ml stock solution, workingsolution prepared by diluting 1:100 to 0.01 mg/ml: 20 microliters added.NRP/Avidin; 0.28 mg/ml stock solution: 90 microliters added.

Fifteen minutes were allowed for diffusion and equilibration after theaddition of the antibody and Con A solutions. Another fifteen minuteswere allowed after the addition of the biotinylated mannatriose andHRP/avidin. To 10 drops of a Dextran Blue solution, at 3.9 mg/ml water,were added 6 drops of fast red TR salt, 2.4 mg/ml, 1 drop of 3% H₂O₂,and 800 ul 50 mM sodium acetate pH 4.5 containing 4 mM MnCl₂ and 4 mMCaCl₂. This solution has been found to show disappearance of absorbanceat 620 nm upon addition of HRP, or the entire antibody-ConA-biotinylated mannatriose-avidin/HRP. At the end of all theseadditions, the total volume in the cuvette was 3.0 ml.

After the addition of the Dextran Blue-based Detection System,absorbance readings at 620 nm were monitored continuously. After thereadings stabilized at 0.40 absorbance units, 500 microliters ofDisplacement Solution were added. This solution was composed ofsaturated alpha methylmannoside in 50 mM sodium acetate pH 4.5containing 4 mM MnCl₂ and 4 mM CaCl₂.

Upon addition of this alpha methylmannoside—the analyte—the absorbancedropped from 0.40 to 0.26 absorbance units. This decrease, 35% is fargreater than the 14% that one would expect based on the dilution from3.0 to 3.5 ml volume, and was reproducible, as seen in severalrepetitions. The majority of the decrease in absorbance was due to theenzymatic action of displaced HRP on the Dextran Blue.

It is apparent that many modifications and variations of the inventionmay be made without departing from the spirit and scope of the presentinvention. It is understood that the invention is not confined to theparticular construction and arrangement herein described, but embracessuch modified forms of it as come within the appended claims. Thespecific embodiments described are given by way of example only and theinvention is limited only by the terms of the appended claims.

1. A method of making a contiguous solid comprising non-lamellar domainswith a plurality of microparticles of matrix dispersed therein, whereineach matrix consists essentially of i. a nanostructured liquid phase ora dehydrated variant thereof, ii. a nanostructured liquid crystallinephase or a dehydrated variant thereof or iii. a combination of (1) ananostructured liquid phase or a dehydrated variant thereof and (2) ananostructured liquid crystalline phase or a dehydrated variant thereofcomprising the steps of: dispersing a volume of said matrix in a form ofsaid nonlamellar material selected from the group consisting ofliquefied form, solution, or fluid precursor, and solidifying saidnonlamellar material by a technique selected from the group consistingof cooling, evaporating a volatile solvent, or implementing a chemicalreactions, wherein said step of solidifying results in production ofsaid contiguous solid comprising non-lamellar domains with a pluralityof microparticles of matrix dispersed therein.
 2. A conglomerate ofcoated particles, each of said coated particles comprising a. aninterior core comprising a matrix consisting essentially of i. at leastone nanostructured liquid phase or a dehydrated variant thereof, ii. atleast one nanostructured liquid crystalline phase or a dehydratedvariant thereof or iii. a combination of (1) at least one nanostructuredliquid phase or a dehydrated variant thereof and (2) at least onenanostructured liquid crystalline phase or a dehydrated variant thereofand b. an exterior coating comprising nonlamellar domains; wherein aplurality of said coated particles are aggregated together to form aconglomerate.
 3. A dispersion of coated microparticles within acontiguous solid comprising a plurality of coated microparticles, eachof said coated microparticles comprising a. an interior core comprisinga matrix consisting essentially of i. at least one nanostructured liquidphase or a dehydrated variant thereof, ii. at least one nanostructuredliquid crystalline phase or a dehydrated variant thereof or iii. acombination of (1) at least one nanostructured liquid phase or adehydrated variant thereof and (2) at least one nanostructured liquidcrystalline phase or a dehydrated variant thereof and b. an exteriorcoating comprising nonlamellar domains; and a contiguous solidcomprising non-lamellar domains in which said plurality of coatedmicroparticles are dispersed.
 4. The dispersion of coated microparticlesof claim 3 wherein non-lamellar domains of said contiguous solid are thesame material as said nonlamellar domains of said exterior coating.
 5. Adispersion of microparticles within a contiguous solid, comprising: aplurality of microparticles, each of said microparticles comprising amatrix consisting essentially of i. at least one nanostructured liquidphase or a dehydrated variant thereof, ii. at least one nanostructuredliquid crystalline phase or a dehydrated variant thereof or iii. acombination of (1) at least one nanostructured liquid phase or adehydrated variant thereof and (2) at least one nanostructured liquidcrystalline phase or a dehydrated variant thereof; and a continuoussolid comprising non-lamellar domains in which said plurality ofmicroparticles are dispersed.
 6. A dispersed particle comprising ananostructured liquid or liquid crystalline matrix with a coatingprecursor localized preferentially at the surface of said particle. 7.The dispersed particle of claim 6, wherein said coating is nonlamellar.8. The dispersed particle of claim 7, wherein said particle becomesencapsulated upon conversion of said coating precursor to a solidnonlamellar coating.
 9. The dispersed particle of claim 6, wherein theprecursor to the coating is a surface-active compound.
 10. The dispersedparticle of claim 9, wherein said coating precursor will be converted toa solid nonlamellar coating by a chemical reaction.
 11. The dispersedparticle of claim 6, wherein said particle is dispersed in an aqueousmedium.
 12. The dispersed particle of claim 6 wherein saidnanostructured liquid crystalline matrix is a reversed liquidcrystalline matrix.
 13. The dispersed particle of claim 12 wherein saidreversed liquid crystalline matrix is a reversed cubic phase liquidcrystalline matrix.
 14. The dispersed particle of claim 6 wherein saidcoating precursor is a salt of N-acetyltryptophan.
 15. A compositioncomprising a divalent ion salt of N-acetyltryptophan, a surfactant, apharmaceutical active compound, and water.
 16. The composition of claim15 wherein the surfactant is a poloxamer.